Small Molecule Inhibitors of the JAK Family of Kinases

ABSTRACT

2-((1r,4r)-4-(imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile compounds, pharmaceutical compositions containing them, methods of making them, and methods of using them including methods for treating disease states, disorders, and conditions mediated by JAK.

FIELD OF THE INVENTION

The present invention relates to certain imidazopyrrolopyridinecompounds, pharmaceutical compositions containing them, methods ofmaking them, and methods of using them as JAK inhibitors and for thetreatment of disease states, disorders, and conditions mediated by JAK.

BACKGROUND

Internal factors, external factors or a combination of both factors cantrigger or be associated with the development of abnormal immuneresponses in the body. Consequently, pathological states develop inwhich constituents, such as substances and tissues, that are normallypresent in the body are subject to such immune response. These statesare generically referred to as immune system diseases. Because thebody's immune system is involved and the damage affects body tissue,such diseases are also referred to as autoimmune diseases. Because suchsystem and tissue are part of the same body, the terms “autoimmunedisease” and “immune system disease” are used here interchangeably,regardless of what triggers the anomalous immune system response.Furthermore, the identity or the mechanism of the underlying immuneproblem is not always clear. See, for example, D. J. Marks, et al.,Crohn's disease: An immune deficiency state, Clinical Reviews in Allergyand Immunology 38(1), 20-30 (2010); J. D. Lalande, et al, Mycobacteriain Crohn's disease: How innate immune deficiency may result in chronicinflammation, Expert Reviews of Clinical Immunology 6(4), 633-41 (2010);J. K. Yamamoto-Furusho, et al., Crohn's disease: Innateimmunodeficiency, World Journal of Gastroenterology, 12(42), 6751-55(2006). As used herein, the term “autoimmune disease” does not excludeconditions whose causes comprise external factors or agents, such asenvironmental or bacterial factors, and internal factors such as geneticsusceptibility. Accordingly, a condition such as Crohn's disease (CD) isreferred to herein as an autoimmune disease, regardless of whether it istriggered by the body itself or by external factors. See, e.g., J. L.Casanova, et al., Revisiting Crohn's disease as a primaryimmunodeficiency of macrophages, J. Exp. Med. 206(9), 1839-43 (2009).

Among the various adverse effects caused by autoimmune diseases, atleast one of the following is typically observed: Damage to, andsometimes destruction of, tissues, and organ alteration that can impactorgan growth and organ function. Examples of autoimmune diseases affectmost major organs, endocrine and exocrine glands, the blood and muscles,and a plurality of systems, such as the digestive, vascular, connectiveand nervous systems. Immunosuppressive treatments are often adopted totreat autoimmune diseases.

Multiple theories are known to explain how autoimmune diseases arise,some focusing on endogenous factors and others also including exogenousfactors. At the molecular level, the Janus kinase/signal transducer andactivator of transcription (JAK/STAT) signaling pathway is considered toplay an important role in transmitting information from extracellularchemical signals to the cell nucleus resulting in regulation of genesthat are involved in cellular activities such as immunity. Cytokines arean example of an extracellular molecule that plays an important role incell signaling. Leukocytes such as neutrophils are recruited bycytokines and chemokines to ultimately cause tissue damage in chronicinflammatory diseases.

The Janus kinase (JAK) family of proteins consists of 4 tyrosinekinases, JAK1, JAK2, JAK3 and Tyk2, which are central to theintracellular signaling of type I and type II cytokine receptors. Theterm JAK refers to either JAK1, JAK2, JAK3 or Tyk2, or any combinationthereof. Each JAK selectively associates with receptor subunits whichdimerize (or multimerize) to form functional receptors. According to J.D. Clark, et al., Discovery and Development of Janus Kinase (JAK)Inhibitors for Inflammatory Diseases, J. Med. Chem. 57(12), 5023-38(2014), “the activation step occurs when a cytokine binds to itsreceptor, inducing a multimerization (dimerization or higher ordercomplexes) of receptor subunits. This brings the JAKs associated witheach subunit proximal to one another, triggering a series ofphosphorylation events ultimately resulting in the phosphorylation andactivation of signal transducers and activators of transcription (STAT)proteins. A phosphorylated STAT dimer then translocates to the nucleusof the cell where it binds to target genes modulating their expression.”Once in the nucleus, STATs regulate gene transcription of numerousmediators in the inflammatory process via binding to specificrecognition sites on DNA. See, for example, J. Med. Chem. 57(12),5023-38 (2014), cited above. Considerable evidence exists demonstratingthe importance for the JAK/STAT pathway in inflammatory, autoimmunediseases and cancer. See, for example, M. Coskun, et al., Involvement ofJAK/STAT signaling in the pathogenesis of inflammatory bowel disease,Pharmacological Research 76, 1-8 (2013); and J. J. O'Shea, et al., JAKsand STATs in immunity, immunodeficiency, and cancer, The New EnglandJournal of Medicine 368, 161-70 (2013).

Inflammatory bowel diseases, including Crohn's disease and ulcerativecolitis (UC), are characterized by recurrent intestinal inflammation,disruption of the epithelial barrier and microbial dysbiosis. Theexcessive inflammatory response in the gastrointestinal tract ismediated by several pro-inflammatory cytokines including TNFα, IFN-γ,IL-1, IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL-17, IL-21, and IL-23that exert their effects on cells of the innate and adaptive immunesystem including T and B lymphocytes, epithelial cells, macrophages anddendritic cells (DC). See, for example, Pharmacological Research 76, 1-8(2013), cited above; S. Danese, et al., JAK inhibition using tofacitinibfor inflammatory bowel disease treatment: A hub for multipleinflammatory cytokines, American Journal of Physiology, Gastrointestinaland Liver Physiology 310, G155-62 (2016); and M. F. Neurath, Cytokinesin inflammatory bowel disease, Nature Reviews Immunology 14, 329-42(2014).

Prevention and/or control of such excessive inflammatory response isdesireable. In light of the mechanism of such response as summarizedabove, JAK inhibition (see illustration in FIG. 1 in the form of anjagged arrow showing a pan-JAK inhibitor striking upon the JAK/STATsignaling pathway and inflammation) is envisaged to prevent or controlexcessive inflammatory response. JAK inhibitors that inhibit a pluralityof such JAK proteins, are referred to here as pan-JAK inhibitors.Examples of therapeutic benefits of such prevention or control have beenseen with tofacitinib, an orally bioavailable pan-JAK inhibitor approvedin the United States for the treatment of rheumatoid arthritis andcurrently in clinical development for ulcerative colitis. In a Phase 2clinical trial, 194 patients with moderate to severe ulcerative colitiswere reportedly evaluated for clinical efficacy. See, e.g., W. J.Sandborn, et al., Tofacitinib, an oral Janus kinase inhibitor, in activeulcerative colitis, The New England Journal of Medicine 367, 616-24(2012). Published information on this trial indicates that patientsreceiving twice a day (BID) doses of 0.5, 3, 10 and 15 mg achievedclinical response rates of 32, 48, 61 and 78%, respectively, compared to42% observed in placebo. It was further reported that the secondary endpoint of clinical remission (Mayo score ≤2) was 13, 33, 48 and 41%compared to 10% observed in placebo. See, e.g., The New England Journalof Medicine 367, 616-24 (2012), cited above. In a Phase 3 UC clinicaltrial, 88 out of 476 patients reportedly achieved clinical remissionfollowing 8 weeks of treatment with tofacitinib (10 mg BID) compared to10 out of 122 patients receiving placebo treatment. See W. J. Sandborn,et al. Efficacy and safety of oral tofacitinib as induction therapy inpatients with moderate-to-severe ulcerative colitis: results from 2phase 3 randomised controlled trials, J. Crohns Colitis 10, S15-S(2016).Reports on Crohn's disease indicate that tofacitinib was also indevelopment for the treatment of CD; however, it was reportedlydiscontinued due to failure to achieve clinical efficacy in a 4week/Phase 2 clinical trial for moderate to severe CD. See W. J.Sandborn, et al., A phase 2 study of tofacitinib, an oral Janus kinaseinhibitor, in patients with Crohn's disease, Clinical gastroenterologyand hepatology: The official clinical practice journal of the AmericanGastroenterological Association 12, 1485-93 e2 (2014). Based onconsulted publicly available literature, it is currently unclear whetherthe tofacitinib failure in CD relates to clinical study design,mechanistic differences between UC and CD or dose-limiting systemicadverse events. See Pharmacological Research 76, 1-8 (2013), citedabove; Clinical gastroenterology and hepatology: the official clinicalpractice journal of the American Gastroenterological Association 12,1485-93 e2 (2014), cited above; and C. J. Menet, et al.,Triazolopyridines as selective JAK1 inhibitors: from hit identificationto GLPG0634, J. Med. Chem. 57, 9323-42 (2014). In light of the featuresof this JAK inhibitor, it is desirable to find additional JAK inhibitorsfor the prevention and/or control of excessive inflammatory response.

Systemic adverse events have been reported with respect to both Phase 2and Phase 3 inflammatory bowel disease (IBD) clinical trials withtofacitinib. See The New England Journal of Medicine 367, 616-24 (2012),cited above; Clinical gastroenterology and hepatology: the officialclinical practice journal of the American GastroenterologicalAssociation 12, 1485-93 e2 (2014), cited above; and J. Panes, et al.Efficacy and safety of oral tofacitinib for induction therapy inpatients with moderate-to-severe Crohn's disease: results of a Phase 2brandomised placebo-controlled trial, J. Crohns Colitis 10, S18-S19(2016). These adverse events include decreased absolute neutrophilcounts (ANC), elevated total cholesterol (low and high-density lipid),intestinal perforation, and infection. Such adverse events areconsistent with those observed following tofacitinib treatment inrheumatoid arthritis (RA) patients (see, for example, J. M. Kremer, etal. The safety and efficacy of a JAK inhibitor in patients with activerheumatoid arthritis: Results of a double-blind, placebo-controlledphase IIa trial of three dosage levels of CP-690,550 versus placebo,Arthritis and Rheumatism 60, 1895-905 (2009)), some of which likelyresult from either JAK2 dependent inhibition of EPO, TPO and colonystimulating factors (csf-2 and GM-CSF (granulocyte macrophage-colonystimulating factor)) and/or JAK1 dependent inhibition of IL-6. See,Arthritis and Rheumatism 60, 1895-905 (2009), cited above; and O. H.Nielsen, et al., Will novel oral formulations change the management ofinflammatory bowel disease? Expert Opinion on Investigational Drugs 25,709-18 (2016).

In reference to FIG. 1, an orally administered medication can inprinciple follow the gastro-intestinal tract from the mouth to theesophagus (1), to the stomach (2) through the duodenum (3) to thejejunum (4), then to the ileum (5), and then to the colon (6). Therelative absorption areas for such various parts are approximately 60%for the jejunum (4), approximately 26% for the ileum (5), andapproximately 13% for the colon (6). Absorption through these variousgastro-intestinal regions can lead to the onset of systemic distributionthat in turn could lead to undesirable side-effects. Thegastro-intestinal tract has a very large surface area. See, for example,H. F. Helander, et al., Surface area of the digestive tract—revisited,Scandinavian Journal of Gastroenterology 49(6), 681-89 (2014); and K. J.Filipski, et al., Intestinal Targeting of Drugs: Rational DesignApproaches and Challenges Current Topics in Medicinal Chemistry 13,776-802 (2013). Such an extensive absorption surface area favorssystemic distribution of substances that can go through the walls of thevarious parts of the intestinal tract and into the blood stream, and inturn have the potential to lead to unwanted side effects of asystemically distributed substance. Systemic distribution is representedby dashed line arrows in FIG. 1 as permeating through the colon wallsfor simplified illustrative purposes, but such distribution is notlimited to the colon walls, for it also can take place through the wallsof other parts of the gastrointestinal tract shown in FIG. 1, such asthose of the small intestine. It is also understood that the dashedarrow lines in FIG. 1 represent systemic distribution beyond thegastrointestinal track as such systemic distribution is known to takeplace in reference to the gastrointestinal track physiology, and thatsuch dashed line arrows simply refer in a schematic illustrative mannerto such systemic distribution. See, for example, Current Topics inMedicinal Chemistry 13, 777-80 (2013), cited above, for a description ofintestinal tissue, transport across the same, and metabolism.

One major reason for attrition in drug candidates is safety andtolerability. See, for example, I. Kola, et al., Can the pharmaceuticalindustry reduce attrition rates? Nature Reviews Drug Discovery 3, 711-5(2004); M. J. Waring, et al., An analysis of the attrition of drugcandidates from four major pharmaceutical companies. Nature Reviews DrugDiscovery 14, 475-86 (2015); M. Hay, et al., Clinical developmentsuccess rates for investigational drugs, Nature Biotechnology 32, 40-51(2014); and M. E. Bunnage, Getting pharmaceutical R&D back on target,Nature Chemical Biology 7, 335-9 (2011). Increasing local tissueconcentrations of compound to the intended target tissue, while limitingexposure to other tissue, can reduce unwanted side effects. See, forexample, V. P. Torchilin, Drug targeting. European Journal ofPharmaceutical Sciences: Official Journal of the European Federation forPharmaceutical Sciences11 Suppl 2, S81-91 (2000). This concept haswidely been accepted for certain diseases and tissues, such as eye (see,for example, R. Gaudana, et al., Ocular drug delivery, The AAPS Journal12, 348-60 (2010)), skin (see, for example, R. Folster-Holst, et al.,Topical hydrocortisone 17-butyrate 21-propionate in the treatment ofinflammatory skin diseases: pharmacological data, clinical efficacy,safety and calculation of the therapeutic index, Die Pharmazie 71,115-21 (2016)), and lung (see, for example, J. S. Patil, et al.,Pulmonary drug delivery strategies: A concise, systematic review, LungIndia: official organ of Indian Chest Society 29, 44-9 (2012)). Similarto these tissue-targeting approaches, increasing intestinal drugconcentrations while limiting unwanted drug levels in other tissue canincrease safety margins. See, for example, I. R. Wilding, et al.,Targeting of drugs and vaccines to the gut, Pharmacology & Therapeutics62, 97-124 (1994); D. Charmot, Non-systemic drugs: a critical review,Current Pharmaceutical Design 18, 1434-45 (2012); and Current Topics inMedicinal Chemistry 13, at 780 (2013), cited above. Tissue-selectivemodulation of targets in the gastrointestinal tissue with compoundsachieving limited systemic exposures can potentially improve thetherapeutic index of such compounds for the treatment of diseases of thegastrointestinal tract including ulcerative colitis and Crohn's disease.See, for example, O. Wolk, et al., New targeting strategies in drugtherapy of inflammatory bowel disease: mechanistic approaches andopportunities, Expert Opin. Drug Deliv. 10(9), 1275-86 (2013). The term“systemic effects” is used herein to refer to systemic exposure and theeffects of any such systemic exposure, even though they are not alwaysthe same.

Because some known JAK inhibitors have adverse effects that areassociated with their systemic effects, it is desirable to find new JAKinhibitors as active substances for the prevention and/or control ofexcessive inflammatory response and whose systemic effects areeliminated or reduced. It is furthermore desireable to find JAKinhibitors with local effects on gastro-intestinal tissues for thetreatment of conditions such as, but not limited to IBD, with reducedsystemic effects. Because of the role played by the various JAKproteins, it is furthermore desirable to find pan-JAK inhibitors.

Intestinal tissue targeting can in principle be pursued according tomultiple strategies. See, for example, Current Topics in MedicinalChemistry 13, at 780-95 (2013), cited above, referring to approachesthat include physicochemical property approaches, transport-mediatedapproaches, prodrug approaches, and formulation and technologyapproaches. It is acknowledged, however, that a “number of challengesand pitfalls exist that are endemic to tissue targeting programs” and inparticular to intestinally targeted compounds, as described in CurrentTopics in Medicinal Chemistry 13, at 795 (2013), cited above.

IBD conditions can extend to multiple parts of the gastrointestinaltract. Even though for simplified illustrative purposes only a colonicdisease site (10) is shown in the descending colon in FIG. 1,inflammatory bowel disease may affect any part of the gastrointestinaltract as is the case with Crohn's disease, or in the rectum and colon,as with ulcerative colitis. See, for example, NIDDK (National Instituteof Diabetes, and Digestive and Kidney Diseases, National Institutes ofHealth, US Department of Health and Human Services,<http://spotidoc.com/doc/71780/crohns-disease-national-digestive-diseases-information>,accessed Nov. 29, 2016. IBD disease sites can be, for example, ileal(ileum-located), ileocolic (affecting portions of the ileum and colon),and colonic (located in the colon, as illustratively shown in thedescending colon in FIG. 1). So, in certain disease scenarios, a drugdelivery along the entire or a large portion of the intestinal tract maybe desirable. In other disease scenarios, it may be desirable toincrease local concentration at any given portion of thegastrointestinal tract. Still in other scenarios, a combination of thesetwo forms of delivery at different sites in the intestinal tract couldbe desirable.

One of such scenarios would focus on the delivery of an active substancethat has limited systemic effects due to limited absorption when passingthrough the gastrointestinal tract as exemplified by the solid linearrows in FIG. 1, while being available to act in extensive portions ofthe gastrointestinal (GI) tract, a feature that is referred to herein as“local GI effects”. Because of reduced systemic effects, a wider rangeof dosages could be evaluated for such substance. It would be furtherdesirable if such active substance had low permeability, so that only asmall amount passes through the intestinal wall into the blood stream tolimit undesirable adverse side effects when it reaches non-targetedareas.

In addition, JAK inhibitors are envisaged as treatment candidates forother diseases. They are envisaged for use in the treatment of ocularconditions including dry eye (B. Colligris, et al., Recent developmentson dry eye disease treatment compounds, Saudi J. Ophthalmol. 28(1),19-30 (2014)), myeloproliferative neoplasms, myeloproliferative diseases(E. J. Baxter, et al., Acquired mutation of the tyrosine kinase JAK2 inhuman myeloproliferative disorders, Lancet 365, 1054-1061 (2005); C.James, et al., A unique clonal JAK2 mutation leading to constitutivesignalling causes polycythaemia vera, Nature 434, 1144-1148 (2005); R.Kralovics, et al., A gain-of-function mutation of JAK2 inmyeloproliferative disorders, N. Engl. J. Med. 352, 1779-1790 (2005); R.L. Levine, et al., Activating mutation in the tyrosine kinase JAK2 inpolycythemia vera, essential thrombocythemia, and myeloid metaplasiawith myelofibrosis, Cancer Cell 7, 387-397 (2005); G. Wernig, et al.,Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of amurine model of JAK2V617F-induced polycythemia vera, Cancer Cell 13,311-320 (2008)), myeloproliferative syndrome, acute myeloid leukemia,systemic inflammatory response syndrome, systemic onset juvenilerheumatoid arthritis, juvenile idiopathic arthritis (H. W. Li, et al.,Effect of miR-19a and miR-21 on the JAK/STAT signaling pathway in theperipheral blood mononuclear cells of patients with systemic juvenileidiopathic arthritis, Exp. Ther. Med. 11(6), 2531-2536 (2016)), type IIIhypersensitivity reactions, type IV hypersensitivity, inflammation ofthe aorta, iridocyclitis/uveitis/optic neuritis, juvenile spinalmuscular atrophy, diabetic retinopathy, diabetic kidney diseaseincluding diabetic nephropathy (F. C. Brosius, et al., JAK inhibition inthe treatment of diabetic kidney disease, Diabetologia 59(8), 1624-7,(2016); C. C. Berthier, et al., Enhanced expression of Januskinase-signal transducer and activator of transcription pathway membersin human diabetic nephropathy, Diabetes 58(2), 469-77, (2009); E. N.Gurzov, et al., The JAK/STAT pathway in obesity and diabetes, FEBS J.283(16), 3002-15 (2016)), microangiopathy, inflammation (M. Kopf, etal., Averting inflammation by targeting the cytokine environment, NatureReviews Drug Discovery 9, 703-718 (2010); J. J. O'Shea, et al., A newmodality for immunosuppression: targeting the JAK/STAT pathway, NatureRev. Drug Discov. 3, 555-564 (2004)), chronic inflammation, inflammatorybowel disease including ulcerative colitis (UC) and Crohn's disease (R.H. Duerr, et al., A genome-wide association study identifies IL23R as aninflammatory bowel disease gene, Science 314, 1461-1463 (2006); M.Coskun, et al., Involvement of JAK/STAT signaling in the pathogenesis ofinflammatory bowel disease, Pharmacol. Res. 76, 1-8 (2013); M. J.Waldner, et al., Master regulator of intestinal disease: IL-6 in chronicinflammation and cancer development, Semin. Immunol. 26(1), 75-9 (2014);S. Danese, et al., JAK inhibition using tofacitinib for inflammatorybowel disease treatment: a hub for multiple inflammatory cytokines, Am.J. Physiol. Gastrointest. Liver Physiol. 310(3), G155-62 (2016); W.Strober, et al., Proinflammatory cytokines in the pathogenesis ofinflammatory bowel diseases, Gastroenterology 140, 1756-1767 (2011)),allergic diseases, vitiligo, atopic dermatitis (R. Bissonnette, et al.,Topical tofacitinib for atopic dermatitis: a phase IIa randomized trial,Br. J. Dermatol. 175(5), 902-911 (2016); W. Amano, et al., JAK inhibitorJTE-052 regulates contact hypersensitivity by downmodulating T cellactivation and differentiation, J. Dermatol. Sci. 84, 258-265 (2016); T.Fukuyama, et al., Topically Administered Janus-Kinase InhibitorsTofacitinib and Oclacitinib Display Impressive Antipruritic andAnti-Inflammatory Responses in a Model of Allergic Dermatitis, J.Pharmacol. Exp. Ther. 354(3), 394-405 (2015)), alopecia areata (A. K.Alves de Medeiros, et al., JAK3 as an Emerging Target for TopicalTreatment of Inflammatory Skin Diseases, PLoS One 11(10) (2016); L.Xing, et al., Alopecia areata is driven by cytotoxic T lymphocytes andis reversed by JAK inhibition, Nat. Med. 20(9), 1043-9 (2014)),dermatitis scleroderma, acute or chronic immune disease associated withorgan transplantation (P. S. Changelian, et al. Prevention of organallograft rejection by a specific Janus kinase 3 inhibitor, Science 302,875-878 (2003); F. Behbod, et al. Concomitant inhibition of Janus kinase3 and calcineurin-dependent signaling pathways synergistically prolongsthe survival of rat heart allografts, J. Immunol, 166, 3724-3732 (2001);S. Busque, et al, Calcineurin-inhibitor-free immunosuppression based onthe JAK inhibitor CP-690,550: a pilot study in de novo kidney allograftrecipients, Am. J. Transplant, 9, 1936-1945 (2009)), psoriaticarthropathy, ulcerative colitic arthropathy, autoimmune bullous disease,autoimmune haemolytic anaemia, rheumatoid arthritis (J. M. Kremer, etal., A randomized, double-blind placebo-controlled trial of 3 doselevels of CP-690,550 versus placebo in the treatment of activerheumatoid arthritis, Arthritis Rheum. 54 (annual meeting abstract), L40(2006); W. Williams, et al, A randomized placebo-controlled study ofINCB018424, a selective Janus kinase1&2 (JAK1&2) inhibitor in rheumatoidarthritis (RA), Arthritis Rheum. 58, S431 (2008); N. Nishimoto, et al.,Study of active controlled monotherapy used for rheumatoid arthritis, anIL-6 inhibitor (SAMURAI): evidence of clinical and radiographic benefitfrom an x ray reader-blinded randomised controlled trial of tocilizumab,Ann. Rheum. Dis. 66(9), 1162-7 (2007)), rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus (A. Goropevsek,et al., The Role of STAT Signaling Pathways in the Pathogenesis ofSystemic Lupus Erythematosus, Clin. Rev. Allergy Immunol. (on-linepre-publication)<http://www.docguide.com/role-stat-signaling-pathways-pathogenesis-systemic-lupus-erythematosus?tsid=5>May 23, 2016; M. Kawasaki, et al., Possible role of the JAK/STATpathways in the regulation of T cell-interferon related genes insystemic lupus erythematosus, Lupus. 20(12), 1231-9 (2011); Y. Furumoto,et al., Tofacitinib ameliorates murine lupus and its associated vasculardysfunction, Arthritis Rheumatol., (on-line pre-publication)<https://www.ncbi.nlm.nih.gov/pubmed/27429362> Jul. 18, 2016)), systemiclupus erythematosus associated lung disease,dermatomyositis/polymyositis associated lung disease, asthma (K. Vale,Targeting the JAK/STAT pathway in the treatment of ‘Th2-high’ severeasthma, Future Med. Chem. 8(4), 405-19 (2016)), ankylosing spondylitis(AS) (C. Thompson, et al., Anti cytokine therapy in chronic inflammatoryarthritis, Cytokine 86, 92-9 (2016)), AS-associated lung disease,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, psoriasis (C. L.Leonardi, et al., Efficacy and safety of ustekinumab, a humaninterleukin-12/23 monoclonal antibody, in patients with psoriasis:76-week results from a randomised, double-blind, placebo-controlledtrial (PHOENIX 1), Lancet 371, 1665-1674 (2008); G. Chan, et al.,Dose-dependent reduction in psoriasis severity as evidence ofimmunosuppressive activity of an oral Jak3 inhibitor in humans, Am. J.Transplant. 6, S87 (2006); K. A. Papp, et al., Efficacy and safety oftofacitinib, an oral Janus kinase inhibitor, in the treatment ofpsoriasis: a phase 2b randomized placebo-controlled dose-ranging study,Br. J. Dermatol. 167, 668-677 (2012); M. Cargill, et al. A large-scalegenetic association study confirms IL12B and leads to the identificationof IL23R as psoriasis-risk genes, Am. J. Hum. Genet. 80, 273-290(2007)), psoriasis type 1, psoriasis type 2, plaque psoriasis, moderateto severe chronic plaque psoriasis, autoimmune neutropaenia, spermautoimmunity, multiple sclerosis (all subtypes, B. M. Segal, et al.,Repeated subcutaneous injections of IL 12/23 p40 neutralising antibody,ustekinumab, in patients with relapsing-remitting multiple sclerosis: aphase II, double-blind, placebo-controlled, randomised, dose-rangingstudy, Lancet Neurol. 7, 796-804 (2008); Z. Yan, et al., Role of theJAK/STAT signaling pathway in regulation of innate immunity inneuroinflammatory diseases, Clin. Immunol. (online pre-publication)<https://www.ncbi.nlm.nih.gov/pubmed/27713030>, accessed Oct. 3, 2016;E. N. Benveniste, et al., Involvement of the janus kinase/signaltransducer and activator of transcription signaling pathway in multiplesclerosis and the animal model of experimental autoimmuneencephalomyelitis, J. Interferon Cytokine Res. 34(8), 577-88 (2014); Y.Liu, et al., Therapeutic efficacy of suppressing the Jak/STAT pathway inmultiple models of experimental autoimmune encephalomyelitis, J.Immunol. 192(1), 59-72 (2014)), acute rheumatic fever, Sjogren'ssyndrome, Sjogren's syndrome/disease associated lung disease (T.Fujimura, et al., Significance of Interleukin-6/STAT Pathway for theGene Expression of REG Iα, a New Autoantigen in Sjogren's SyndromePatients, in Salivary Duct Epithelial Cells, Clin. Rev. Allergy Immunol.(online pre-publication) <https://www.ncbi.nlm.nih.gov/pubmed/27339601>Jun. 24, 2016), autoimmune thrombocytopaenia, neuroinflammationincluding Parkinson's disease (Z. Yan, et al., Oct. 3, 2016, citedabove). JAK inhibitors have been reported as having therapeuticapplications in cancer treatment in addition to inflammatory diseases.(S. J. Thomas, et al., The role of JAK/STAT signaling in thepathogenesis, prognosis and treatment of solid tumors, British J. Cancer113, 365-71 (2015); A. Kontzias, et al., Jakinibs: A new class of kinaseinhibitors in cancer and autoimmune disease, Current Opinion inPharmacology, 12(4), 464-70 (August 2012); M. Pesu, et al., Therapeutictargeting of JANUS kinases, Immunological Reviews, 223, 132-42 (June2008); P. Norman, Selective JAK inhibitors in development for rheumatoidarthritis, Expert Opinion on Investigational Drugs, 23(8), 1067-77(August 2014)). In addition, JAK inhibitors could be useful in theprevention of colorectal cancer because inflammation reduction in thecolon could lead to cancer prevention in such organ.

BRIEF SUMMARY OF THE INVENTION

This invention relates to the following compounds:

-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide;-   2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide;-   N-(2-Cyanoethyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)acetamide;-   N-(2-Cyano-2-methylpropyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((1-hydroxycyclobutyl)methyl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1-methyl-1H-pyrazol-4-yl)acetamide;-   N-(4-(Cyanomethyl)bicyclo[2.2.1]heptan-1-yl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((1-hydroxycyclopropyl)methyl)acetamide;    and

pharmaceutically acceptable salts of such compounds, and combinations ofthem.

The term “compounds of the invention” and “compound of the invention” isintended to encompass at least one compound selected from the abovegroup of compounds, whether in a solvent-free form or in any one ofhydrated and/or solvated forms as illustrated herein.

Embodiments of the present invention relate to compounds, pharmaceuticalcompositions containing them, methods of making and purifying them,methods of using them as JAK inhibitors and methods for using them inthe treatment of disease states, disorders, and conditions mediated byJAK.

Embodiments of this invention exhibit pan-JAK inhibition effects withlocal GI effects and low or negligible systemic effects. Furthermore,embodiments of this invention with such features can be orallyadministered.

An additional embodiment of the invention is a method of treating asubject suffering from or diagnosed with a disease, disorder, or medicalcondition mediated by JAK using compounds of the invention or activeagents of the invention.

Additional embodiments, features, and advantages of the invention willbe apparent from the following detailed description and through practiceof the invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1

Schematic diagram of part of the human gastrointestinal tract, shown asa not-at-scale stretched rendering. The duodenum (3), jejunum (4), andileum (5) (all schematically shown) form the small intestine after thestomach (2) and esophagus (1). The large intestine comprises the colon(6), in turn including the cecum (7) and appendix (not shown), ascendingcolon, transverse colon, descending colon, sigmoid colon (loop in thesame not shown), and rectum (11). The transverse colon is the portioncomprised between the right (8) and left (9) colonic flexures, theascending colon extends from the cecum (7) to the right colonic flexure(8), and the descending colon extends from the left colonic flexure (9)to the rectum (11). Various distribution patterns are illustrated inreference to the colon for convenience, but they can also refer to otherparts of the gastrointestinal tract. Systemic distribution isrepresented by dashed line arrows in FIG. 1 as permeating through thecolon walls for simplified illustrative purposes, but such distributionis not limited to the colon walls, for it also can take place throughthe walls of other parts of the gastrointestinal tract shown in FIG. 1,such as those of the small intestine. Distribution with some tissuepenetration is represented by solid line arrows in FIG. 1 as penetratingthe colon tissue for simplified illustrative purposes, but suchpenetration is not limited to the colon tissue, for it also can takeplace in the tissue of other parts of the gastrointestinal tract shownin FIG. 1, such as the tissue of the small intestine. The effect of anembodiment of a JAK inhibitor according to this invention isillustratively shown as disrupting the JAK/STAT signaling pathway thatotherwise would lead to inflammation associated with an inflammatorybowel disease (“IBD”), such as Crohn's disease or ulcerative colitis. Byway of example, but not as a limitation, a disease site isillustratively shown as a colonic disease site (10) in the descendingcolon.

FIG. 2

Schematic diagram showing the preparation/interconversion of embodimentsof compound Ex. 1. Embodiments 19-36, 38 and 39 were obtained fromembodiment 1s, and embodiments 37 and 40-53 were obtained fromembodiment 19, as symbolized in this figure by the dashed line arrow andthe legend “19-53” in the box shown in the same.

FIG. 3

Overlay of high throughput X-ray powder diffraction (HT-XRPD) patternsfor the following embodiments of compound Ex. 1, from bottom to top: 1s,2 (obtained by equilibration at room temperature in 1,4-dioxane), 3b(obtained by thermocycling in cyclohexanone), 1b+4 (obtained by coolingcrystallization at μL scale in methanol/water (50/50, v/v), 5 (obtainedby thermocycling in chloroform), 6 (obtained by cooling crystallizationat mL scale in acetonitrile), 7 (obtained of 1 s+7, in turn obtained bysolvent equilibration in heptane), 7 (obtained by desolvation of 1 s+7,in turn obtained by solvent equilibration in heptane), 8 (obtained bydesolvation of embodiment 5 by cycling differential scanningcalorimetry)), and 9 (obtained by desolvation of embodiment 2 by cyclingdifferential scanning calorimetry).

FIG. 4

Overlay of high throughput X-ray powder diffraction (HT-XRPD) patternsfor the following embodiments of compound Ex. 1, from bottom to top: 1s(starting material), 1a (obtained after exposure to accelerated agingconditions (AAC) (40° C. and 70% relative humidity) several forms ofsamples of embodiment 1s), 1b (obtained by solvent equilibration at roomtemperature in toluene), 1c (obtained by cooling crystallization at μLscale in ethyl acetate/1,4-dioxane (50/50, v/v)), 1d (obtained bycooling crystallization at μL scale in acetonitrile/chloroform (50/50,v/v)), 1e (obtained by cooling crystallization at μL scale in ethylacetate/1,4-dioxane (50/50, v/v)), if (obtained by solvent equilibrationat room temperature in p-xylene), 1g (obtained by solvent equilibrationat 50° C. in anisole), 1h (obtained by cooling crystallization at μLscale in p-xylene).

FIG. 5

Overlay of high throughput X-ray powder diffraction (HT-XRPD) patternsfor the following embodiments of compound Ex. 1, from bottom to top: 1s,3b (obtained by thermocycling in cyclohexanone), 3c (obtained by coolingcrystallization at μL scale in 1,4-dioxane), 3d (obtained by coolingcrystallization at μL screen in tetrahydrofuran), and 3e (obtained bythermocycling in isobutanol).

FIG. 6

HR-XRPD diffractograms of embodiment 1s in its initial form (“1s”),after a four-day exposure to 40° C. and 70% relative humidity (“1s 70RH”), and after a four-day exposure to 25° C. and 100% relative humidity(embodiment 10 or “10”).

FIG. 7A

X-ray powder diffraction (XRPD) pattern of embodiment 11.

FIG. 7B

X-ray powder diffraction (XRPD) pattern of embodiment 12.

FIG. 7C

X-ray powder diffraction (XRPD) pattern of embodiment 13.

FIG. 7D

X-ray powder diffraction (XRPD) pattern of embodiment 14.

FIG. 7E

X-ray powder diffraction (XRPD) pattern of embodiment 11b.

FIG. 8

Overlay of X-ray powder diffraction (XRPD) patterns for the followingembodiments of compound Ex. 1, from bottom to top: embodiment 17,embodiment 18, embodiment 15 and embodiment 16.

FIG. 9

Modulated DSC (“mDSC”) profile for embodiment 19 showing a glasstransition point (T_(g)) at 115.3° C. (“Rev” in the ordinate axis labelrefers to “reversible”).

FIG. 10A

TGA (thermogravimeteric analysis) of embodiment 18 showing a 6.5% w/wloss between 30° C. and 170° C.

FIG. 10B

DSC (differential scanning calorimetry) of embodiment 18 showing anendotherm of 52.8 J/g between 45° C. and 90° C., an endotherm of 31.0J/g at 140.6° C., an exotherm of 24.3 J/g at 168.8° C., and an endothermof 31.3 J/g at 200.0° C.

FIG. 11

Overlay of X-ray powder diffraction (XRPD) patterns for the followingembodiments of compound Ex. 1, from bottom to top: embodiment 20 andembodiment 21.

FIG. 12A

TGA of embodiment 17 showing a 4.2% w/w loss between 30° C. and 100° C.

FIG. 12B

DSC of embodiment 17 showing an endotherm of 90.3 J/g between 45° C. and100° C., an endotherm of 35.5 J/g at 143.8° C., an endotherm of 1.6 J/gat 168.3° C., an exotherm of 3.8 J/g at 178° C., and an endotherm of 9.2J/g at 200.0° C.

FIG. 13

Overlay of X-ray powder diffraction (XRPD) patterns for the followingembodiments of compound Ex. 1, from bottom to top: embodiment 31,embodiment 30, embodiment 17, embodiment 29, embodiment 16, embodiment26, embodiment 25, embodiment 18, embodiment 24, embodiment 23,embodiment 27 and embodiment 22.

FIG. 14

Overlay of X-ray powder diffraction (XRPD) patterns for the followingembodiments of compound Ex. 1, from bottom to top: embodiment 32,embodiment 33, embodiment 23, embodiment 34, embodiment 35, embodiment36, embodiment 25, embodiment 38, embodiment 17, embodiment 39 andembodiment 28.

FIG. 15

Overlay of X-ray powder diffraction (XRPD) patterns for the followingembodiments of compound Ex. 1, from bottom to top: embodiment 46,embodiment 45, embodiment 44, embodiment 43, embodiment 42, embodiment41, embodiment 40 and embodiment 37.

FIG. 16

Overlay of X-ray powder diffraction (XRPD) patterns for the followingembodiments of compound Ex. 1, from bottom to top: embodiment 53,embodiment 52, embodiment 51, embodiment 50, embodiment 49, embodiment48 and embodiment 47.

FIG. 17A

TGA of embodiment 11 showing a 4.7% w/w loss between 155° C. and 185° C.

FIG. 17B

DSC of embodiment 11 showing a first endotherm of 57.8 J/g at 167.8° C.due to solvent loss and a second endotherm of 90.8 J/g at 194.5° C. dueto sample melt.

FIG. 18

Gravimetric Vapor Sorption (GVS) isotherm plot of embodiment 11 showinga mass change of 0.66% between 0-90% RH. The mass change on the ordinateaxis is in reference to the mass of the starting sample.

FIG. 19A

TGA of embodiment 6 showing weight loss at temperatures above 260° C.,which weight loss is interpreted as being associated with sampledegradation.

FIG. 19B

DSC of embodiment 6 showing an endotherm of 95.8 J/g at 194.4° C. due tosample melt.

FIG. 20A

TGA of embodiment 8 showing a 1.4% w/w loss between 40° C. and 240° C.,which corresponds to a loss of 0.07 mol of 1,4-dioxane.

FIG. 20B

DSC of embodiment 8 showing an endotherm of 58.6 J/g at 199.7° C. due tosample melt.

FIG. 21A

TGA of embodiment 2 showing a 7.4% w/w loss between 75° C. and 110° C.,a 11.9% w/w loss between 110° C. and 130° C., a 2.0% w/w loss between130° C. and 165° C., and a 2.5% w/w loss between 165° C. and 210° C.

FIG. 21B

DSC of embodiment 2 showing an endotherm of 86.2 J/g at 92.8° C., anendotherm of 11.1 J/g at 111.5° C., an endotherm of 45.5 J/g at 149.0°C., an exotherm of 20.6 J/g at 165.2° C., an endotherm of 3.7 J/g at177.1° C., an endotherm of 43.0 J/g at 200.2° C., and an endotherm of29.3 J/g at 220.6° C.

FIG. 22A

TGA of embodiment 9.

FIG. 22B

DSC of embodiment 9 showing an endotherm of 104.4 J/g at 221.8° C.

FIG. 23A

TGA of embodiment 16 showing a 5.2% w/w loss between 30° C. and 105° C.

FIG. 23B

DSC of embodiment 16 showing an endotherm of 48.4 J/g between 35° C. and90° C., an endotherm of 41.8 J/g at 147.0° C., an endotherm of 1.0 J/gat 166.6° C., an exotherm of 4.4 J/g at 180.7° C., and an endotherm of7.7 J/g at 201.1° C.

FIG. 24

X-ray powder diffraction (XRPD) of embodiment 11 (labeled “11”) andX-ray powder diffraction (XRPD) of embodiment 11 after the variabletemperature (VT)-XRPD experiment (labeled “11 post VT”), and X-raypowder diffraction (XRPD) of embodiment 6.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “including”, “containing” and “comprising” areused in their open, non-limiting sense.

Any formula given herein is intended to represent compounds havingstructures depicted by the structural formula as well as certainvariations or forms. Certain structures may exist as tautomers.Additionally, an amorphous form, hydrates, solvates, polymorphs andpseudopolymorphs of such compounds of this invention, and mixturesthereof, are also envisaged as parts of this invention. Embodiments ofthis invention are in a solvent-free form or in any one of hydratedand/or solvated forms as illustrated herein.

Reference to a compound herein stands for a reference to any one of: (a)the actually recited form of such compound, and (b) any of the forms ofsuch compound in the medium in which the compound is being consideredwhen named. For example, reference herein to a compound such as R—COOH,encompasses reference to any one of, for example, R—COOH_((s)),R—COOH_((sol)), and R—COO⁻ _((sol)). In this example, R—COOH_((s))refers to the solid compound, as it could be for example in a tablet orsome other solid pharmaceutical composition or preparation;R—COOH_((sol)) refers to the undissociated form of the compound in asolvent; and R—COO⁻ _((sol)) refers to the dissociated form of thecompound in a solvent, such as the dissociated form of the compound inan aqueous environment, whether such dissociated form derives fromR—COOH, from a salt thereof, or from any other entity that yields R—COO⁻upon dissociation in the medium being considered. In another example, anexpression such as “exposing an entity to compound of formula R—COOH”refers to the exposure of such entity to the form, or forms, of thecompound R—COOH that exists, or exist, in the medium in which suchexposure takes place. In still another example, an expression such as“reacting an entity with a compound of formula R—COOH” refers to thereacting of (a) such entity in the chemically relevant form, or forms,of such entity that exists, or exist, in the medium in which suchreacting takes place, with (b) the chemically relevant form, or forms,of the compound R—COOH that exists, or exist, in the medium in whichsuch reacting takes place. In this regard, if such entity is for examplein an aqueous environment, it is understood that the compound R—COOH isin such same medium, and therefore the entity is being exposed tospecies such as R—COOH_((aq)) and/or R—COO⁻ _((aq)), where the subscript“(aq)” stands for “aqueous” according to its conventional meaning inchemistry and biochemistry. A carboxylic acid functional group has beenchosen in these nomenclature examples; this choice is not intended,however, as a limitation but it is merely an illustration. It isunderstood that analogous examples can be provided in terms of otherfunctional groups, including but not limited to hydroxyl, basic nitrogenmembers, such as those in amines, and any other group that interacts ortransforms according to known manners in the medium that contains thecompound. Such interactions and transformations include, but are notlimited to, dissociation, association, tautomerism, solvolysis,including hydrolysis, solvation, including hydration, protonation, anddeprotonation. No further examples in this regard are provided hereinbecause these interactions and transformations in a given medium areknown by any one of ordinary skill in the art.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number in an enriched form. Examples of isotopesthat can be incorporated into compounds of the invention in a form thatexceeds natural abundances include isotopes of hydrogen, carbon,nitrogen, and oxygen such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, and ¹⁷O,respectively. Such isotopically labeled compounds are useful inmetabolic studies (preferably with ¹⁴C), reaction kinetic studies (with,for example deuterium (i.e., D or ²H); or tritium (i.e., T or ³H)),detection or imaging techniques [such as positron emission tomography(PET) or single-photon emission computed tomography (SPECT)] includingdrug or substrate tissue distribution assays, or in radioactivetreatment of patients. In particular, an ¹⁸F or ¹¹C labeled compound maybe particularly preferred for PET or SPECT studies. Further,substitution with heavier isotopes such as deuterium (i.e., ²H) mayafford certain therapeutic advantages resulting from greater metabolicstability, for example increased local in vivo half-life or reduceddosage requirements. Isotopically labeled compounds of this inventioncan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures that have an H memberin different positions may be in equilibrium while satisfying valencyrules. For example, enols and ketones are tautomers because they arerapidly interconverted by treatment with either acid or base.

When referring to any formula given herein, the selection of aparticular moiety from a list of possible species for a specifiedvariable is not intended to define the same choice of the species forthe variable appearing elsewhere. In other words, where a variableappears more than once, the choice of the species from a specified listis independent of the choice of the species for the same variableelsewhere in the formula, unless stated otherwise.

By way of a first example on substituent terminology, if substituent S¹_(example) is one of S₁ and S₂, and substituent S² _(example) is one ofS₃ and S₄, then these assignments refer to embodiments of this inventiongiven according to the choices S¹ _(example) is S₁ and S² _(example) isS₃; S¹ _(example) is S₁ and S² _(example) is S₄; S¹ _(example) is S₂ andS² _(example) is S₃; S¹ _(example) is S₂ and S² _(example) is S₄; andequivalents of each one of such choices. The shorter terminology “S¹_(example) is one of S₁ and S₂, and S² _(example) is one of S₃ and S₄”is accordingly used herein for the sake of brevity, but not by way oflimitation. The foregoing first example on substituent terminology,which is stated in generic terms, is meant to illustrate the varioussubstituent assignments described herein.

Furthermore, when more than one assignment is given for any member orsubstituent, embodiments of this invention comprise the variousgroupings that can be made from the listed assignments, takenindependently, and equivalents thereof. By way of a second example onsubstituent terminology, if it is herein described that substituentS_(example) is one of S₁, S₂, and S₃, this listing refers to embodimentsof this invention for which S_(example) is S₁; S_(example) is S₂;S_(example) is S₃; S_(example) is one of S₁ and S₂; S_(example) is oneof S₁ and S₃; S_(example) is one of S₂ and S₃; S_(example) is one of S₁,S₂ and S₃; and S_(example) is any equivalent of each one of thesechoices. The shorter terminology “S_(example) is one of S₁, S₂, and S₃”is accordingly used herein for the sake of brevity, but not by way oflimitation. The foregoing second example on substituent terminology,which is stated in generic terms, is meant to illustrate the varioussubstituent assignments described herein.

The following JAK inhibitors are illustrative embodiments of theinvention:

-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide;-   2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide;-   N-(2-Cyanoethyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)acetamide;-   N-(2-Cyano-2-methylpropyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((1-hydroxycyclobutyl)methyl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1-methyl-1H-pyrazol-4-yl)acetamide;-   N-(4-(Cyanomethyl)bicyclo[2.2.1]heptan-1-yl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide;-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide;    and-   2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((1-hydroxycyclopropyl)methyl)acetamide.

Additional embodiments of the invention are pharmaceutically acceptablesalts of compounds given above.

Additional embodiments of the invention are pharmaceutical compositionseach comprising an effective amount of at least one of the compoundsgiven above or a pharmaceutically acceptable salt thereof.

A “pharmaceutically acceptable salt” is a salt of a compound that isnon-toxic, biologically tolerable, or otherwise biologically suitablefor administration to the subject. See, generally, S. M. Berge, et al.,“Pharmaceutical Salts”, J. Pharm. Sci. 66, 1-19 (1977), and Handbook ofPharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth,Eds., Wiley-VCH and VHCA, Zurich, 2002. Compounds of the invention maypossess a sufficiently acidic group, a sufficiently basic group, or bothtypes of functional groups, and accordingly react with a number ofinorganic or organic bases, and inorganic and organic acids, to form apharmaceutically acceptable salt. Examples of pharmaceuticallyacceptable salts include sulfates, pyrosulfates, bisulfates, sulfites,bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,propionates, decanoates, caprylates, acrylates, formates, isobutyrates,caproates, heptanoates, propiolates, oxalates, malonates, succinates,suberates, sebacates, fumarates, maleates, butyne-1,4-dioates,hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates,sulfonates, xylenesulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, methane-sulfonates, propanesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

If the compound of the invention contains at least one basic nitrogen,the desired pharmaceutically acceptable salt may be prepared by anysuitable method available in the art, for example, treatment of the freebase with an inorganic acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, sulfamic acid, nitric acid, boric acid, andphosphoric acid, or with an organic acid, such as acetic acid,phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbicacid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid,valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid,glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, apyranosidyl acid, such as glucuronic acid or galacturonic acid, analpha-hydroxy acid, such as mandelic acid, citric acid, or tartaricacid, an amino acid, such as aspartic acid or glutamic acid, an aromaticacid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, orcinnamic acid, a sulfonic acid, such as laurylsulfonic acid,p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, anycompatible mixture of acids such as those given as examples herein, andany other acid and mixture thereof that are regarded as equivalents oracceptable substitutes in light of the ordinary level of skill in thistechnology.

Not all the embodiments of pharmaceutically acceptable salts ofcompounds according to this invention may be equally suitable for theirdevelopment, for compounds that are sufficiently weakly basic (e.g., pKaof about 4) might not form sufficiently stable salts for developmentpurposes. See, e.g., G. A. Stephenson, et al., J. Pharm. Sciences100(5), 1607-17 (2011) “Physical stability of salts of weak bases in thesolid state”. Some embodiments of this invention are envisaged toencompass co-crystallized forms of a compound according to thisinvention with a suitable co-crystal former. Design and properties ofco-crystals for pharmaceutical use and methods of making andcharacterizing them have been given in, for example, N. Shan, et al.,Drug Discovery Today, 13(9/10), 440-46 (2008) “The role of cocrystals inpharmaceutical science”; N. Qiao, et al., Intl. J. Pharmaceutics, 419,1-11 (2011) “Pharmaceutical cocrystals: An overview”; R. Thakuria, etal., Intl. J. Pharmaceutics, 453, 101-25 (2013) “Pharmaceuticalcocrystals and poorly soluble drugs”.

The compounds of the invention, including their pharmaceuticallyacceptable salts, whether alone or in combination, (collectively,“active agent” or “active agents”) are useful as JAK inhibitors in themethods of the invention. Such methods for modulating JAK activitycomprise exposing JAK to an effective amount of at least one chemicalcompound of the invention.

In some embodiments, the JAK inhibitor is used in a subject diagnosedwith or suffering from a disease, disorder, or medical conditionmediated through JAK activity, such as those described herein. Symptomsor disease states are intended to be included within the scope of“diseases, disorders or medical conditions.”

Accordingly, the invention relates to methods of using the active agentsdescribed herein to treat subjects diagnosed with or suffering from adisease, disorder, or medical condition mediated through JAK. The term“treat” or “treating” as used herein is intended to refer toadministration of an active agent or composition of the invention to asubject for the purpose of affecting a therapeutic or prophylacticbenefit through modulation of JAK. Treating includes reversing,ameliorating, alleviating, inhibiting the progress of, lessening theseverity of, reducing, or preventing a disease, disorder, or condition,or one or more symptoms of such disease, disorder or condition mediatedthrough modulation of JAK activity. The term “subject” refers to amammalian patient in need of such treatment, such as a human. The term“inhibitors” or “inhibitor” refers to compounds that decrease, prevent,inactivate, desensitize or down-regulate JAK expression or activity.

Embodiments of this invention provide JAK inhibitors for the preventionand/or control of excessive inflammatory response. Embodiments of JAKinhibitors according to this invention are pan-JAK inhibitors.

Unless indicated otherwise, the term “JAK inhibitor physico-chemicalproperties” refers to the corresponding named properties as follows:

as given in the description for compounds Ex. 1-12, in the case of molarmasses;

as determined according to the respective definitions, in the case ofnumbers of H bond donors, acceptors and rotatable bonds; and

as measured in reference to Table 1a, column 2, in case of plasmaconcentrations, and Table 7, columns 3 and 4, in case of the A-Bpermeability coefficients in the presence of P-gp inhibitor and B-Apermeability coefficients.

Embodiments of this invention provide methods of inhibiting JAK,comprising exposing a JAK receptor to a JAK inhibitor that ischaracterized by having the following JAK inhibitor physico-chemicalproperties: a plasma concentration in the range from about 0.1 ng/mL toabout 60 ng/mL, c Log P in the range from about 0.1 to about 2.8, A-Bpermeability coefficients in the presence of a P-gp inhibitor in therange from about 0.1 to about 2.5, B-A permeability coefficients in therange from about 0.5 to about 20, tPSA in the range from about 85 toabout 120.

In other embodiments of methods of inhibiting JAK according to thisinvention, the plasma concentration is in the range from about 10 ng/mLto about 20 ng/mL.

In other embodiments of methods of inhibiting JAK according to thisinvention, c Log P is in the range from about 0.8 to about 1.4.

In other embodiments of methods of inhibiting JAK according to thisinvention, the A-B permeability coefficient in the presence of a P-gpinhibitor is in the range from about 0.6 to about 1.5.

In other embodiments of methods of inhibiting JAK according to thisinvention, the B-A permeability coefficient is in the range from about0.5 to about 5.

In other embodiments of methods of inhibiting JAK according to thisinvention, the tPSA is in the range from about 100 to about 120.

Further embodiments of this invention provide methods of inhibiting JAK,comprising exposing a JAK receptor to a JAK inhibitor that is furthercharacterized by having the following JAK inhibitor physico-chemicalproperties: A molar mass in the range from about 300 g mol⁻¹ to about500 g mol⁻¹, a number of hydrogen bond donors in the range from about 2to about 3, a number of hydrogen bond acceptors in the range from about4 to about 5, and a number of rotatable bonds in the range from about 3to about 6, in addition to the plasma concentrations, c log P values,permeability coefficients, and tPSA values described above formethodologies of inhibiting JAK according to this invention.

In other embodiments of methods of inhibiting JAK according to thisinvention, the molar mass is in the range from about 340 g mol⁻¹ toabout 430 g mol⁻¹.

In other embodiments of methods of inhibiting JAK according to thisinvention, the number of rotatable bonds is in the range from about 5 toabout 6.

Embodiments of this invention provide methods for treating inflammationin the gastrointestinal tract of a subject, comprising administering toa subject a pharmaceutically effective amount of a JAK inhibitor that ischaracterized by having the following JAK inhibitor physico-chemicalproperties: A plasma concentration in the range from about 0.1 ng/mL toabout 60 ng/mL, c Log P in the range from about 0.1 to about 2.8, A-Bpermeability coefficients in the presence of a P-gp inhibitor in therange from about 0.1 to about 2.5, B-A permeability coefficients in therange from about 0.5 to about 20, tPSA in the range from about 85 toabout 120.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, the plasmaconcentration is in the range from about 10 ng/mL to about 20 ng/mL.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, c Log P is in therange from about 0.8 to about 1.4.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, the A-B permeabilitycoefficient is in the presence of a P-gp inhibitor is in the range fromabout 0.6 to about 1.5.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, the B-A permeabilitycoefficient is in the range from about 0.5 to about 5.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, the tPSA is in therange from about 100 to about 120.

Further embodiments of this invention provide methods for treatinginflammation in the gastrointestinal tract of a subject wherein the JAKinhibitor physico-chemical properties are further characterized byhaving the following JAK inhibitor physico-chemical properties: A molarmass in the range from about 300 g mol¹ to about 500 g mol⁻¹, a numberof hydrogen bond donors in the range from about 2 to about 3, a numberof hydrogen bond acceptors in the range from about 4 to about 5, and anumber of rotatable bonds in the range from about 3 to about 6, inaddition to the plasma concentrations, c Log P values, permeabilitycoefficients, and tPSA values described above for methodologies oftreating inflammation according to this invention.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, the molar mass is inthe range from about 350 g mol⁻¹ to about 430 g mol⁻¹.

In other embodiments of methods of treating inflammation in thegastrointestinal tract according to this invention, the number ofrotatable bonds is in the range from about 5 to about 6.

Embodiments of JAK inhibitors according to this invention have thefollowing JAK physico-chemical properties: a plasma concentration in therange from about 0.1 ng/mL to about 60 ng/mL, a c Log P in the rangefrom 0.1 to about 2.8, an A-B permeability coefficient in the presenceof a P-gp inhibitor in the range from about 0.1 to about 2.5, a B-Apermeability coefficient in the range from about 0.5 to about 20, and atPSA in the range from about 85 to about 120.

Further embodiments of JAK inhibitors according to this invention have aplasma concentration is in the range from about 10 ng/mL to about 20ng/mL.

Further embodiments of JAK inhibitors according to this invention have cLog P values in the range from about 0.8 to about 1.4.

Further embodiments of JAK inhibitors according to this invention haveA-B permeability coefficient in the presence of a P-gp inhibitor in therange from about 0.6 to about 1.5.

Further embodiments of JAK inhibitors according to this invention haveB-A permeability coefficient in the range from about 0.5 to about 5.

Further embodiments of JAK inhibitors according to this invention havetPSA values in the range from about 100 to about 120.

Other embodiments of JAK inhibitors according to this invention have thefollowing JAK inhibitor physico-chemical properties: A molar mass in therange from about 300 g mol⁻¹ to about 500 g mol⁻¹, a number of hydrogenbond donors in the range from about 2 to about 3, a number of hydrogenbond acceptors in the range from about 4 to about 5, and a number ofrotatable bonds in the range from about 3 to about 6 in addition to theplasma concentrations, c Log P values, permeability coefficients, andtPSA values described above for JAK inhibitors according to thisinvention.

Further embodiments of JAK inhibitors according to this invention have amolar mass is in the range from about 350 g mol⁻¹ to about 430 g mol⁻¹.

Further embodiments of JAK inhibitors according to this invention have anumber of rotatable bonds is in the range from about 5 to about 6.

In treatment methods according to the invention, an effective amount ofat least one active agent according to the invention is administered toa subject suffering from or diagnosed as having such a disease,disorder, or medical condition. An “effective amount” means an amount ordose sufficient to generally bring about the desired therapeutic orprophylactic benefit in patients in need of such treatment for thedesignated disease, disorder, or medical condition. Effective amounts ordoses of the active agents of the present invention may be ascertainedby methods such as modeling, dose escalation studies or clinical trials,and by taking into consideration factors, e.g., the mode or route ofadministration or drug delivery, the pharmacokinetics of the agent, theseverity and course of the disease, disorder, or condition, thesubject's previous or ongoing therapy, the subject's health status andresponse to drugs, and the judgment of the treating physician. For a70-kg human, an illustrative range for a suitable dosage amount is fromabout 1 to 1000 mg/day in single or multiple dosage units.

Embodiments of this invention are new JAK inhibitors as activesubstances for the prevention and/or control of excessive inflammatoryresponse and whose systemic effects are eliminated or reduced. Furtherembodiments of this invention are JAK inhibitors with local effects ongastro-intestinal tissues for the treatment of conditions such as, butnot limited to, IBD, without causing systemic effects or with suchsystemic effects acceptably reduced.

Embodiments of this invention are low permeability JAK inhibitors.Further embodiments of this invention are JAK inhibitors that haveaqueous solubility.

Once improvement of the patient's disease, disorder, or condition hasoccurred, the dose may be adjusted for preventive or maintenancetreatment. For example, the dosage or the frequency of administration,or both, may be reduced as a function of the symptoms, to a level atwhich the desired therapeutic or prophylactic effect is maintained. Ofcourse, if symptoms have been alleviated to an appropriate level,treatment may cease. Patients may, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

In addition, the compounds of the invention are envisaged for use alone,in combination with one or more of other compounds of this invention, orin combination with additional active ingredients in the treatment ofthe conditions discussed below. The additional active ingredients may beco-administered separately with at least one compound of the invention,with active agents of the invention or included with such an agent in apharmaceutical composition according to the invention. In anillustrative embodiment, additional active ingredients are those thatare known or discovered to be effective in the treatment of conditions,disorders, or diseases mediated by JAK activity, such as another JAKinhibitor or a compound active against another target associated withthe particular condition, disorder, or disease. The combination mayserve to increase efficacy (e.g., by including in the combination acompound potentiating the potency or effectiveness of an agent accordingto the invention), decrease one or more side effects, or decrease therequired dose of the active agent according to the invention.

When referring to inhibiting the target, an “effective amount” means anamount sufficient to affect the activity of at least one of the JAKfamily of proteins. Measuring the activity of the target may beperformed by analytical methods.

The active agents of the invention are envisaged for use, alone or incombination with one or more additional active ingredients, to formulatepharmaceutical compositions of the invention. A pharmaceuticalcomposition of the invention comprises an effective amount of at leastone active agent in accordance with the invention.

Pharmaceutically acceptable excipients commonly used in pharmaceuticalcompositions are substances that are non-toxic, biologically tolerable,and otherwise biologically suitable for administration to a subject,such as an inert substance, added to a pharmacological composition orotherwise used as a vehicle, carrier, or diluent to facilitateadministration of an agent and that is compatible therewith. Examples ofsuch excipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils, and polyethylene glycols.

Delivery forms of the pharmaceutical compositions containing one or moredosage units of the active agents may be prepared using pharmaceuticallyacceptable excipients and compounding techniques known or that becomeavailable to those of ordinary skill in the art. The compositions may beadministered in the inventive methods by a suitable route of delivery,e.g., oral, parenteral, rectal, topical, or ocular routes, or byinhalation.

The preparation may be in the form of tablets, capsules, sachets,dragees, powders, granules, lozenges, powders for reconstitution, liquidpreparations, or suppositories. The compositions may be formulated forany one of a plurality of administration routes, such as intravenousinfusion, subcutaneous injection, topical administration, or oraladministration. Preferably, the compositions may be formulated for oraladministration.

For oral administration, the active agents of the invention can beprovided in the form of tablets, capsules, or beads, or as a solution,emulsion, or suspension. To prepare the oral compositions, the activeagents may be formulated to yield a dosage of, e.g., for a 70-kg human,from about 1 to 1000 mg/day in single or multiple dosage units as anillustrative range.

Oral tablets may include the active ingredient(s) mixed with compatiblepharmaceutically acceptable excipients such as diluents, disintegratingagents, binding agents, lubricating agents, sweetening agents, flavoringagents, coloring agents and preservative agents. Suitable inert fillersinclude sodium and calcium carbonate, sodium and calcium phosphate,lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol, and the like. Liquid oral excipients includeethanol, glycerol, water, and the like. Starch, polyvinyl-pyrrolidone(PVP), sodium starch glycolate, microcrystalline cellulose, and alginicacid are disintegrating agents. Binding agents may include starch andgelatin. The lubricating agent, if present, may be magnesium stearate,stearic acid or talc. If desired, the tablets may be coated with amaterial such as glyceryl monostearate or glyceryl distearate to delayabsorption in the gastrointestinal tract, or may be coated with anenteric coating. Additional coating that may be used include coatingsthat are designed to release the compound or active agent as a functionof time, pH or bacterial content.

Capsules for oral administration include hard and soft gelatin or(hydroxypropyl)methyl cellulose capsules. To prepare hard gelatincapsules, active ingredient(s) may be mixed with a solid, semi-solid, orliquid diluent. Soft gelatin capsules may be prepared by mixing theactive ingredient with an oil such as peanut oil or olive oil, liquidparaffin, a mixture of mono and di-glycerides of short chain fattyacids, polyethylene glycol 400, or propylene glycol. Liquids for oraladministration may be in the form of suspensions, solutions, emulsionsor syrups or may be lyophilized or presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid compositions may optionally contain: pharmaceutically-acceptableexcipients such as suspending agents (for example, sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethylcellulose,carboxymethylcellulose, aluminum stearate gel and the like); non-aqueousvehicles, e.g., oil (for example, almond oil or fractionated coconutoil), propylene glycol, ethyl alcohol, or water; preservatives (forexample, methyl or propyl p-hydroxybenzoate or sorbic acid); wettingagents such as lecithin; and, if desired, flavoring or coloring agents.

The active agents of this invention may also be administered by non-oralroutes. For example, compositions may be formulated for rectaladministration as a suppository, enema or foam. For parenteral use,including intravenous, intramuscular, intraperitoneal, or subcutaneousroutes, the agents of the invention may be provided in sterile aqueoussolutions or suspensions, buffered to an appropriate pH and isotonicityor in parenterally acceptable oil. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. Such forms may bepresented in unit-dose form such as ampules or disposable injectiondevices, in multi-dose forms such as vials from which the appropriatedose may be withdrawn, or in a solid form or pre-concentrate that can beused to prepare an injectable formulation. Illustrative infusion dosesrange from about 1 to 1000 μg/kg/minute of agent admixed with apharmaceutical carrier over a period ranging from several minutes toseveral days.

For topical administration, the agents may be mixed with apharmaceutical carrier at a concentration of about 0.01% to about 20% ofdrug to vehicle, preferably 0.1% to 10%. Another mode of administeringthe agents of the invention may utilize a patch formulation to effecttransdermal delivery.

Active agents may alternatively be administered in methods of thisinvention by inhalation, via the nasal or oral routes, e.g., in a sprayformulation also containing a suitable carrier.

In a further embodiment, the invention is directed to a method oftreating a subject suffering from or diagnosed with a disease, disorder,or medical condition mediated by JAK, comprising administering to thesubject in need of such treatment an effective amount of the activeagent.

In certain embodiments of the inventive method, the disease, disorder,or medical condition is an inflammatory bowel disease, such as Crohn'sdisease and ulcerative colitis.

Other embodiments of this invention provide for a method for modulatingJAK activity, including when such kinase is in a subject, comprisingexposing JAK to an effective amount of at least one compound selectedfrom compounds of the invention.

The compounds of the invention are useful as JAK inhibitors that can bedosed orally and specifically distribute to intestinal tissue whilemaintaining low systemic exposures. This is in contrast to most knownJAK inhibitors which are dosed orally and distribute to many tissues dueto the fact that they have extensive systemic exposure.

Table 1a and Table 1b show results of in vivo experiments. These resultscomprise plasma and colon tissue concentrations for fifteen compoundsthat had been administered to mice as described in Protocols 1, 2 or 3.Plasma and colon concentration results were obtained by followingProtocol 1 using venipuncture of dorsal metatarsal vein bleed forCompounds (B), (C), and Examples 6 and 11. Plasma and colonconcentration results were obtained by following Protocol 2 usingretro-orbital bleed for Compounds (A), and Examples 1, and 3-5 andProtocol 2 using venipuncture of the dorsal metatarsal vein for Examples2, 7-10, and 12. The results of Protocols 1 and 2 are shown in Table 1a.Plasma and colon concentration results were obtained by followingProtocol 3 for Examples 1, 3 and 4. The results of Protocol 3 are shownin Table 1b. These protocols are described below under the heading Invivo Studies.

TABLE 1a Results of In Vivo Experiments After p.o. Dosing - MeanConcentration of Test Compounds Colon Concentration After p.o. PlasmaConcentration After p.o. Dosing (ng/mL) Dosing (ng/g) Time = 0.5 h Time= 2 h Time = 4 h Time = 4 h Test Standard Standard Standard StandardCompound Mean* Deviation Mean* Deviation Mean* Deviation Mean* DeviationA 347.0 78.5 69.1 40.8 84.5 25.5 895.0 260.6 B 352.7 85.7 66.3 26.2 11.33.7 6076.7 3125.8 C 547.0 71.4 130.2 63.7 16.7 5.9 7776.7 3500.2 Ex. 113.4 1.5 6.1 3.7 3.3 1.2 8591.7 10245.7 Ex. 2 24.5 3.6 4.2 1.8 1.3 0.17600.0 983.6 Ex. 3 41.4 15.1 3.9 0.7 1.5** *** 2147.2 1821.6 Ex. 4 12.91.6 7.5 2.8 3.3 1.5 4448.3 989.3 Ex. 5 31.9 5.1 8.8 1.7 6.0 1.2 5328.3986.0 Ex. 6 18.8 20.6 3.0 0.9 1.7^(#) ^(##) 11706.7 11305.2 Ex. 7 47.03.8 9.6 4.4 5.0 1.2 12008.3 9461.1 Ex. 8 43.1 8.7 5.4 0.6 2.6 0.6 7396.73037.3 Ex. 9 15.1 1.8 6.2 4.5 3.9 0.9 7683.3 230.9 Ex. 10 26.6 4.0 3.21.0 3.1 0.7 3005.0 1347.2 Ex. 11 1.6** *** {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 4785.0 1059.9 Ex. 12 15.6 8.74.2 1.4 2.3 1.0 5885.0 3154.1 *Mean calculated from the values obtainedfrom three mice unless otherwise noted. **Mean was calculated withvalues obtained from two mice as the values obtained from the thirdmouse were below the lower limit of quantitation. *** No standarddeviation calculated as the mean was calculated from only two values.^(#)Mean given as the value obtained from one mouse as the valuesobtained from the second and third mice were below the lower limit ofquantitation. ^(##)No standard deviation calculated in light of note #in this table. {circumflex over ( )} Mean was not calculated as thevalues for all three mice were below the lower limit of quantitation.{circumflex over ( )}{circumflex over ( )} No standard deviationcalculated in light of note {circumflex over ( )} in this table.

TABLE 1b Results of In Vivo Experiments After i.c. Dosing - MeanConcentration of Test Compounds Colon Concentration After i.c. PlasmaConcentration After i.c. Dosing (ng/mL) Dosing (ng/g) Time = 0.5 h Time= 2 h Time = 4 h Time = 4 h Test Standard Standard Standard StandardCompound Mean* Deviation Mean* Deviation Mean* Deviation Mean* DeviationEx. 1 2.5^(#) ^(##) {circumflex over ( )} {circumflex over( )}{circumflex over ( )} {circumflex over ( )} {circumflex over( )}{circumflex over ( )} 681.0 437.0 Ex. 3 1.5^(#) ^(##) {circumflexover ( )} {circumflex over ( )}{circumflex over ( )} {circumflex over( )} {circumflex over ( )}{circumflex over ( )} 227.8 254.1 Ex. 4 3.8***** 2.5^(#) ^(##) {circumflex over ( )} {circumflex over ( )}{circumflexover ( )} 26.1^(#) ^(##) *Mean calculated from the values obtained fromthree mice unless otherwise noted. **Mean was calculated with valuesobtained from two mice as the values obtained from the third mouse werebelow the lower limit of quantitation. *** No standard deviationcalculated as the mean was calculated from only two values. ^(#)Meangiven as the value obtained from one mouse as the values obtained fromthe second and third mice were below the lower limit of quantitation.^(##)No standard deviation calculated in light of note # in this table.{circumflex over ( )} Mean was not calculated as the values for allthree mice were below the lower limit of quantitation. {circumflex over( )}{circumflex over ( )} No standard deviation calculated in light ofnote {circumflex over ( )} in this table.

Compounds (A)-(C) are the following reference compounds that have beendisclosed in WO2013/007765 or WO2011/086053 for their use as inhibitorsof Janus kinases:

Compounds Ex. 1-12 in Tables 1a and 1b are embodiments of this inventiongiven in the respective Examples.

As evinced in Table 1a, colon concentrations for compounds Ex. 1-12 werefound to be much higher than the respective plasma concentrations, with[colon (4 h)]: [plasma (0.5 h)] concentration ratios ranging from about52 to about 3,000. In contrast, such ratios for compounds (A)-(C) rangedfrom about 3 to about 17. Table 1b also provides supportive data thatExamples 1, 3 and 4 have low systemic exposures after i.c. dosing. Thecontrast between properties of embodiments of this invention withrespect to reference compounds is much more accentuated when thecomparison is referred to the 4 h plasma concentration values. In thisregard, the [colon (4 h)]: [plasma (4 h)] concentration ratios forcompounds Ex. 1-12 range from about 888 to about 6886. In contrast, suchratios for compounds (A)-(C) range from about 11 to about 538. Thesecolon-to-plasma concentration ratios are indicative of compounds Ex.1-12 having low systemic effects at any time post oral dose, whilecompounds (A)-(C) have comparatively high systemic effects. This is anunexpected finding of local GI effects for compounds Ex. 1-12.

As shown in Table 4, the enzymatic activity of compounds Ex. 1-12 wasmeasured to determine activity for each individual enzyme. For allcompounds tested, there was measured inhibition of enzyme activity,demonstrating that these compounds are pan-JAK inhibitors. The datagiven in this table for compounds (A)-(C) also demonstrate inhibition ofenzyme activity for all JAK proteins by these compounds.

As shown in Table 5, the cellular activities of compounds Ex. 1-12 wereassessed in peripheral blood mononuclear cell (PBMC) using stimuli IL-2,IFN-α, and GM-CSF and measuring inhibition of phosphorylation of STAT5,STAT4, and STAT5, respectively. For all compounds tested, there wasmeasured inhibition of STAT phosphorylation with all three stimuli.

As shown in Table 6, the solubilities of compounds Ex. 1-12 weremeasured in simulated gastric fluid (“SGF”) and simulated intestinalfluid (“SIF”). All compounds tested showed measurable solubility above400 μM in SGF, and in the range of 81 μM to above 400 μM with SIF. Asshown in the same table, these solubility data were comparable to thesolubilities of compounds (A)-(C).

As shown in Table 7, the permeability of compounds (A)-(C) and Ex. 1-12was measured using MDCK-MDR1 cell line with and without elacridar, aP-gp inhibitor. All compounds demonstrated low permeability forapical-to-basolateral transport measurements, with and without P-gpinhibitor (elacridar). The permeability coefficient values for compounds(A)-(C) and Ex. 1-12 were low and comparable for apical-to-basolateraltransport without elacridar (for all such compounds) and with elacridar(for compounds (B)-(C) and compounds Ex. 1-12) (columns 2 and 3 in suchtable), but the basolateral-to-apical permeability coefficients forcompounds (A)-(C) were greater than those for most of the compounds Ex.1-12, as shown in column 4 of the same table. In reference to columns 3and 4 in Table 7, most of compounds Ex. 1-12 have lowapical-to-basolateral permeability coefficients measured in the presenceof elacridar (column 3), and also low basolateral-to-apical permeabilitycoefficients (column 4). These two features characterize such compoundsas being low permeability compounds. The same characterization cannot bemade for compounds (A)-(C), whose basolateral-to-apical permeabilitycoefficients (column 4) are greater than those for most of the compoundsEx. 1-12. Efflux ratios given in the same table for compounds (A)-(C)and Ex. 1-12 show that all these compounds are P-gp substrates.

There is no known reference teaching or suggestion indicating that themarked lack of systemic effects for embodiments of this invention incomparison with those of reference compounds (A)-(C) can be inferredand/or predicted on the basis of structural comparisons or otherfeatures of compounds (A)-(C) such as those discussed in reference toTables 4, 6 and 7 This is so even though reference compounds (A)-(C)present structural similarities of certain moieties with similarmoieties of embodiments of this invention.

In addition, there is no known reference teaching or suggestionindicating that the low permeability feature for embodiments of thisinvention in comparison with those of reference compounds (A)-(C) can beinferred and/or predicted on the basis of structural comparisons.

The following specific examples are provided to further illustrate theinvention and various embodiments.

In obtaining the compounds described in the examples below and thecorresponding analytical data, the following experimental and analyticalprotocols were followed unless otherwise indicated.

Unless otherwise stated, reaction mixtures were magnetically stirred atroom temperature (rt). Where solutions are “dried,” they are generallydried over a drying agent such as Na₂SO₄ or MgSO₄. Where mixtures,solutions, and extracts were “concentrated”, they were typicallyconcentrated on a rotary evaporator under reduced pressure.

Thin-layer chromatography was performed using Merck silica gel 60 F₂₅₄2.5 cm×7.5 cm, 250 μm or 5.0 cm×10.0 cm, 250 μm pre-coated silica gelplates.

Normal-phase flash column chromatography (FCC) was performed on silicagel (SiO₂) eluting with 2 M NH₃ in MeOH/DCM, unless otherwise noted.

Mass spectra (MS) were obtained on an Agilent series 1100 MSD usingelectrospray ionization (ESI) in positive mode unless otherwiseindicated. Calculated (calcd.) mass corresponds to the exact mass.

Nuclear magnetic resonance (NMR) spectra were obtained on Bruker modelDRX spectrometers. The format of the ¹H NMR data below is: chemicalshift in ppm downfield of the tetramethylsilane reference (multiplicity,coupling constant J in Hz, integration).

Chemical names were generated by either ChemDraw (CambridgeSoft,Cambridge, Mass.) or ACD/Name Version 9 (Advanced Chemistry Development,Toronto, Ontario, Canada). By way of example, the designation (1r,4r)refers to the trans orientation around the cyclohexyl ring as generatedusing the naming function of Chemdraw Ultra Pro 14.0.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value.

Whenever a yield is given as a percentage, such yield refers to a massof the entity for which the yield is given with respect to the maximumamount of the same entity that could be obtained under the particularstoichiometric conditions. Reagent concentrations that are given aspercentages refer to mass ratios, unless indicated differently.

Experiments such as TGA, DSC, GVS typically show slight variability inthe data presented based on the individual samples that are beinganalyzed and slight variations in hydration and/or amount of solventpresent.

The TGA plots for individual embodiments are shown in terms oftemperature in ° C. on the X-axis and weight loss in % on the Y-axis.

The DSC plots for individual embodiments are shown in terms oftemperature in ° C. on the X-axis and heat flow in W/g on the Y-axis.The DSC heating rate was 10° C./min. Integrations of endothermic andexothermic events provide the energy absorbed (for an endothermic event)or energy released (for an exothermic event) in J/g. Dashed lines showngoing across the trace represent the area that was integrated.

In the figures where the term “Exo Up” is present, an endothermic eventis reflected by a curve that goes down and an exotherm event isreflected by a curve that goes up.

Some diffractograms have been presented in an overlay arrangement ofdiffractograms that are separated by spacings to allow visualization.Each of the diffractograms is referenced to a zero relative intensitythat is the intersection of each of such diffractograms with theordinate axis or to the lowest relative intensity reading of each ofsuch diffractograms.

Figures that display a plurality of XPRD patterns for any singleembodiment reflect different patterns obtained for samples of suchembodiment that were nevertheless prepared with the same method indifferent solvents.

Abbreviations and acronyms used herein include the following as shownbelow:

Abbreviations and Acronyms Defined

Acronym Term AAC Accelerated aging conditions (40° C. and 70% RH) ACNAcetonitrile aq Aqueous br Broad cLogP Calculated logP DCMDichloromethane DIPEA, DIEA, Diisopropylethylamine or Hunig's base DMADimethylacetamide DMF N,N-Dimethylformamide DMPU1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone DMSO Dimethylsulfoxide DSC Differential Scanning Calorimetry EtOAc, or EA EthylAcetate EtOH Ethanol ESI Electrospray ionization FCC Normal-phase silicagel flash column chromatography g Gram(s) GVS Gravimetric Vapor Sorptionh Hour(s) HPLC High-pressure liquid chromatography HR-XRPD Highresolution X-ray powder diffraction HT-XRPD High throughput X-ray powderdiffraction IPA isopropanol i.c. Intra-colonic Hz Hertz LCMS Liquidchromatography and mass spectrometry M Molar mDSC Modulated DifferentialScanning Calorimetry m/z Mass to charge ratio MeOH Methanol mgMilligram(s) min Minute(s) mL Milliliter(s) μL Microliter(s) mmolMillimole(s) MTBE Methyl tert-butyl ether MS Mass spectrometry NMRNuclear magnetic resonance p.o. per os or by mouth ppm Parts per millionPTFE polytetrafluoroethylene PyBOPBenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphatePyBrOP Bromotripyrrolidinophosphonium hexafluorophosphate RH Relativehumidity R_(t) Retention time Rt or RT Room temperature TFATrifluoroacetic acid TGA Thermogravimeteric Analysis THF TetrahydrofuranTLC Thin layer chromatography tPSA Topological polar surface area XRPDX-ray powder diffraction

Intermediate 1 synthesis and characterization:

2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile

Step A: tert-butyl N-[(1r,4r)-4-(Hydroxymethyl)cyclohexyl]carbamate. Toa 20-L 4-necked round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed(1r,4r)-4-[[(tert-butoxy)carbonyl]amino]cyclohexane-1-carboxylic acid(1066 g, 4.38 mol, 1.00 equiv) and THF (10 L). This was followed by thedropwise addition of BH₃-Me₂S (10 M, 660 mL) at −10° C. over 1 h. Theresulting solution was stirred for 3 h at 15° C. This reaction wasperformed three times in parallel and the reaction mixtures werecombined. The reaction was then quenched by the addition of methanol (2L). The resulting mixture was concentrated under vacuum. This resultedin of tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (3000g, 99.6%) as a white solid. MS (ESI): mass calcd. for C₁₂H₂₃NO₃, 229.32;m/z found, 215.2 [M-tBu+MeCN+H]⁺; ¹H NMR: (300 MHz, CDCl₃): δ 4.40 (s,1H), 3.45 (d, J=6.3 Hz, 2H), 3.38 (s, 1H), 2.05-2.02 (m, 2H), 1.84-1.81(m, 2H), 1.44 (s, 11H), 1.17-1.01 (m, 4H).

Step B: tert-butylN-[(1r,4r)-4-[(Methanesulfonyloxy)methyl]cyclohexyl]carbamate. To a 20 L4-necked round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed tert-butylN-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (1000 g, 4.36 mol, 1.00equiv.), dichloromethane (10 L), pyridine (1380 g, 17.5 mol, 4.00equiv.). This was followed by the dropwise addition of MsCl (1000 g,8.73 mol, 2.00 equiv.) at −15° C. The resulting solution was stirredovernight at 25° C. This reaction was performed in parallel for 3 timesand the reaction mixtures were combined. The reaction was then quenchedby the addition of 2 L of water. The water phase was extracted withethyl acetate (1×9 L). The organic layer was separated and washed with 1M HCl (3×10 L), NaHCO₃ (saturated aq.) (2×10 L), water (1×10 L) andbrine (1×10 L). The mixture was dried over anhydrous sodium sulfate,filtered and concentrated under vacuum. This resulted in of tert-butylN-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (3300 g,82%) as a white solid. LC-MS: MS (ESI): mass calcd. for C₁₃H₂₅NO₅S,307.15; m/z found 292.1, [M-tBu+MeCN+H]⁺; ¹H NMR: (300 MHz, CDCl₃): δ4.03 (d, J=6.6 Hz, 2H), 3.38 (s, 1H), 3.00 (s, 3H), 2.07-2.05 (m, 2H),1.87-1.84 (m, 2H), 1.72-1.69 (m, 1H), 1.44 (s, 9H), 1.19-1.04 (m, 4H).

Step C: tert-butyl N-[(1r,4r)-4-(Cyanomethyl)cyclohexyl]carbamate. To a10 L 4-necked round-bottom flask, was placed tert-butylN-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (1100 g,3.58 mol, 1.00 equiv.), DMSO (5500 mL) and NaCN (406 g, 8.29 mol, 2.30equiv.). The resulting mixture was stirred for 5 h at 90° C. Thisreaction was performed in parallel 3 times and the reaction mixtureswere combined. The reaction was then quenched by the addition of 15 L ofwater/ice. The solids were collected by filtration. The solids werewashed with water (3×10 L). This resulted in tert-butylN-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (2480 g, 97%) as a whitesolid. MS (ESI): mass calcd. for C₁₃H₂₂N₂O₂, 238.17; m/z found 224[M-tBu+MeCN+H]⁺; ¹H NMR: (300 MHz, CDCl₃): δ 4.39 (s, 1H), 3.38 (s, 1H),2.26 (d, J=6.9 Hz, 2H), 2.08-2.04 (m, 2H), 1.92-1.88 (m, 2H), 1.67-1.61(m, 1H), 1.44 (s, 9H), 1.26-1.06 (m, 4H).

Step D: 2-[(1r,4r)-4-Aminocyclohexyl]acetonitrile hydrochloride. To a10-L round-bottom flask was placed tert-butylN-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (620 g, 2.60 mol, 1.00equiv.), and 1,4-dioxane (2 L). This was followed by the addition of asolution of HCl in 1,4-dioxane (5 L, 4 M) dropwise with stirring at 10°C. The resulting solution was stirred overnight at 25° C. This reactionwas performed for 4 times and the reaction mixtures were combined. Thesolids were collected by filtration. The solids were washed with1,4-dioxane (3×3 L), ethyl acetate (3×3 L) and hexane (3×3 L). Thisresulted in 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride(1753 g, 96%) as a white solid. MS (ESI): mass calcd. for C₈H₁₄N₂,138.12; m/z found 139.25, [M+H]⁺; ¹H NMR: (300 MHz, DMSO-d₆): δ 8.14 (s,3H), 2.96-2.84 (m, 1H), 2.46 (d, J=6.3 Hz, 2H), 1.98 (d, J=11.1 Hz, 2H),1.79 (d, J=12.0 Hz, 2H), 1.64-1.49 (m, 1H), 1.42-1.29 (m, 2H), 1.18-1.04(m, 2H).

Step E:2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile.To a 1000 mL round bottom flask containing2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride (29.10 g, 166.6mmol) was added DMA (400 mL). The resulting suspension was treated with4-chloro-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (51.53 g,152.6 mmol), followed by DIPEA (63.0 mL, 366 mmol). The reaction mixturewas placed under N₂ and heated at 80° C. for 4 h. The crude reactionmixture was cooled to room temperature and slowly poured into avigorously stirred 2 L flask containing 1.6 L water. The resultingsuspension was stirred for 15 minutes at room temperature, then filteredand dried for 16 h in a vacuum oven with heating at 70° C. to providethe title compound (63.37 g, 95%) as a yellow solid. MS (ESI): masscalcd. for C₂₁H₂₁N₅₀₄S, 439.1; m/z found, 440.1 [M+H]⁺. ¹H NMR (500 MHz,CDCl₃): δ 9.10 (s, 1H), 8.99 (d, J=7.8 Hz, 1H), 8.23-8.15 (m, 2H),7.66-7.59 (m, 2H), 7.56-7.49 (m, 2H), 6.67 (d, J=4.2 Hz, 1H), 3.95-3.79(m, 1H), 2.38 (d, J=6.2 Hz, 2H), 2.32-2.21 (m, 2H), 2.08-1.98 (m, 2H),1.88-1.76 (m, 1H), 1.60-1.32 (m, 4H).

Intermediate 2 synthesis and characterization:

2-((1r,4r)-4-((5-Amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile

2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile(Intermediate 1, 58.60 g, 133.3 mmol) was dissolved in THF/MeOH (1:1,4800 mL). The mixture was passed through a continuous-flow hydrogenationreactor (10% Pd/C), such as a Thales Nano H-Cube®, at 10 mL/min with100% hydrogen (atmospheric pressure, 80° C.), then the solution wasconcentrated to provide the product as a purple solid. The solid wastriturated with EtOAc (400 mL) and then triturated again with MeOH (200mL) then filtered and dried under vacuum to provide the title compound(50.2 g, 91.9% yield). MS (ESI): mass calcd. for C₂₁H₂₃N₅₀₂S, 409.2; m/zfound, 410.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.10-8.03 (m, 2H), 7.76(s, 1H), 7.51-7.43 (m, 1H), 7.43-7.34 (m, 3H), 6.44 (d, J=4.2 Hz, 1H),4.61 (d, J=8.5 Hz, 1H), 3.65-3.51 (m, 1H), 2.74 (s, 2H), 2.26 (d, J=6.4Hz, 2H), 2.19-2.05 (m, 2H), 1.97-1.86 (m, 2H), 1.76-1.59 (m, 1H),1.33-1.12 (m, 4H).

Intermediate 3 synthesis and characterization:

Ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate

To a 1 L round bottom flask containing a stir bar and2-((1r,4r)-4-((5-amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile(Intermediate 2, 58.31 g, 142.4 mmol) was added ethyl3-ethoxy-3-iminopropanoate (60.51 g, 309.3 mmol), followed by EtOH (600mL, dried over 3 Å molecular sieves for 48 h). A reflux condenser wasattached to the reaction flask, the reaction was purged with N₂, and washeated at 90° C. for 9 h. The reaction mixture was cooled to roomtemperature and left to stand for 30 h where the product crystallizedout as brown needles. The solids were broken up with a spatula and thereaction mixture was transferred to a 2 L flask. Water (1.4 L) was addedslowly via separatory funnel with vigorous stirring. After addition ofthe water was complete, the suspension was stirred for 30 minutes. Thebrown needles were isolated by filtration and then dried by pulling airthrough the filter for 1 h. The product was transferred to a 500 mLflask and treated with EtOAc (200 mL). A small quantity of seed crystalswere added, which induced the formation of a white solid precipitate.The suspension was stirred for 30 minutes at room temperature, filtered,rinsed with EtOAc (25 mL), and dried under vacuum to provide the productas a white solid (48.65 g, 68% yield). MS (ESI): mass calcd. forC₂₆H₂₇N₅O₄S, 505.2; m/z found, 506.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ8.85 (s, 1H), 8.28-8.19 (m, 2H), 7.84 (d, J=4.0 Hz, 1H), 7.61-7.53 (m,1H), 7.52-7.43 (m, 2H), 6.84 (d, J=4.1 Hz, 1H), 4.32 (s, 1H), 4.20 (q,J=7.1 Hz, 2H), 4.09 (s, 2H), 2.44 (d, J=6.2 Hz, 2H), 2.40-2.27 (m, 2H),2.16 (d, J=13.3 Hz, 2H), 2.12-1.96 (m, 3H), 1.54-1.38 (m, 2H), 1.27 (t,J=7.1 Hz, 3H).

Intermediate 4 synthesis and characterization:

Sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate

To a solution of ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 3, 9.50 g, 18.8 mmol) in MeOH (30 mL) and THF (30 mL) wasadded aq sodium hydroxide (56.4 mL, 56.4 mmol, 1 M) and was stirred atroom temperature for 14 hours. The solvent was removed under reducedpressure at room temperature to provide the title compound (7 g) as abrown solid, which was used in the next step without furtherpurification. MS (ESI): mass calcd. for C₁₈H₁₈ N₅NaO₂, 359.1; m/z found,337.9 [M+H-Na]⁺. ¹H NMR (400 MHz, CD₃OD) δ 8.52-8.47 (m, 1H), 7.85-7.81(m, 2H), 7.46-7.41 (m, 4H), 6.85-6.81 (m, 1H), 4.60-4.46 (m, 1H), 3.96(s, 2H), 2.59-2.49 (m, 4H), 2.19-2.05 (m, 6H), 1.56-1.43 (m, 2H) (a 1:1mixture of the title compound and benzenesulfonic acid).

Example 1 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide

Step A:2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide.To ensure dry starting material, ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 3) was heated under vacuum at 50° C. for 18 h prior to thereaction. In a 1 L flask, ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 3, 52.585 g, 104.01 mmol) was suspended in DMA (50 mL).1-Amino-2-methylpropan-2-ol (50 mL) was added and the reaction washeated to 110° C. for 45 minutes, then to 125° C. for 5 hours. Thereaction was cooled to room temperature and diluted with EtOAc (800 mL).The organic layer was extracted three times with a solution ofwater/brine wherein the solution was made up of 1 L water plus 50 mLbrine. The aqueous layers were back extracted with EtOAc (2×600 mL). Thecombined organic layers were dried over anhydrous MgSO₄, concentrated todryness, and then dried for 3 days under vacuum to provide the titlecompound (65.9 g, 98% yield) as a yellow foam. The product was taken tothe next step with no further purification. MS (ESI): mass calcd. forC₂₈H₃₂N₆O₄S, 548.22; m/z found, 549.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃): δ8.76 (s, 1H), 8.26-8.19 (m, 2H), 7.84 (d, J=4.1 Hz, 1H), 7.60-7.53 (m,1H), 7.50-7.44 (m, 2H), 6.84 (d, J=4.2 Hz, 1H), 4.76-4.61 (m, 1H), 3.97(s, 2H), 3.45 (s, 1H), 3.27 (d, J=5.9 Hz, 2H), 2.41 (d, J=6.5 Hz, 2H),2.38-2.25 (m, 2H), 2.23-2.12 (m, 2H), 2.09-1.94 (m, 4H), 1.48 (qd,J=13.6, 4.0 Hz, 2H), 1.21 (s, 6H).

Step B:2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide.2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide(65.90 g, 102.1 mmol) was added to a 1 L flask containing a stir bar.1,4-dioxane (300 mL) was added, followed by aq KOH (3 M, 150 mL). Thereaction was heated at 80° C. for 2 h. The reaction was cooled to roomtemperature and the solvent volume was reduced to about 200 mL on arotovap. The residue was treated with a solution of water/brine (100mL/100 mL), then extracted with 10% MeOH in CH₂Cl₂ (2×1 L). The organiclayers were combined, dried over anhydrous MgSO₄, and concentrated todryness to provide a yellow solid. The solid was suspended in CH₂Cl₂(200 mL), stirred vigorously for 30 minutes, and then collected byfiltration. The solid was rinsed with CH₂Cl₂ (100 mL), dried by pullingair through the filter, and then further dried under vacuum at roomtemperature for 16 h to provide the title compound (41.59 g, 89% yield)as a white solid. MS (ESI): mass calcd. for C₂₂H₂₈N₆O₂, 408.23; m/zfound, 409.2 [M+H]⁺. ¹H NMR (600 MHz, DMSO-d₆): δ 11.85 (s, 1H), 8.50(s, 1H), 8.21-8.10 (m, 1H), 7.49-7.43 (m, 1H), 6.74-6.65 (m, 1H),4.53-4.42 (m, 2H), 4.07 (s, 2H), 3.08 (d, J=6.0 Hz, 2H), 2.58 (d, J=6.1Hz, 2H), 2.41-2.28 (m, 2H), 2.09-1.92 (m, 5H), 1.42-1.31 (m, 2H), 1.09(s, 6H). The synthesis and active compound characterization of each ofthe embodiments of this invention are provided herein in the form ofexamples. Due to the crystal structure of some of the embodiments ofthis invention, polymorph screening may be pursued to furthercharacterize specific forms of any such compound. This is illustrated ina non-limiting manner for compound Ex. 1 by the example under theheading polymorph screening. Tests reported herein concerning compoundEx. 1 were performed with such compound in a form given by embodiment 1sas described in the polymorph screening example below.

Example 2 synthesis and characterization:

2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile

Step A:2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)-6-(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile.2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile(Intermediate 1, 23.3 g, 53.0 mmol) was added into a 1 L round-bottomedflask containing a magnetic stir-bar followed by the addition of DMSO(200 mL) and methanol (200 mL). 1H-Imidazole-4-carbaldehyde (8.56 g,89.1 mmol) was added as a solid, followed by the addition of sodiumhydrosulfite (32.7 g, 188 mmol) as a solution in water (100 mL). Thereaction vessel was equipped with a reflux condenser and heated to 90°C. in a heating block for 15 h. The reaction mixture was then cooled toroom temperature and added to a flask containing water (2000 mL) withstirring, which resulted in formation of a white precipitate. Themixture was stirred for 30 minutes and the solids were collected byfiltration. The solids were dried by pulling air through the filter for6 h and then further dried in a vacuum oven heating at 60° C. for 3 daysto provide the title compound (22.7 g, 88% yield) as a yellow solid. MS(ESI): mass calcd. for C₂₅H₂₃N₇O₂S, 485.16; m/z found, 486.1 [M+H]⁺. ¹HNMR (400 MHz, CDCl₃): δ 8.74 (s, 1H), 8.29 (s, 1H), 8.20-8.11 (m, 2H),8.04-7.96 (m, 2H), 7.76-7.68 (m, 1H), 7.68-7.60 (m, 2H), 7.19 (d, J=4.2Hz, 1H), 5.56 (s, 1H), 2.58 (d, J=6.3 Hz, 2H), 2.38-2.24 (m, 2H), 2.07(s, 1H), 1.98 (d, J=10.8 Hz, 5H), 1.35 (q, J=12.3 Hz, 2H).

Step B:2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile.The title compound was prepared using conditions analogous to thosedescribed in Example 1, Step B using2-((1r,4r)-4-(2-(1H-imidazol-4-yl)-6-(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile(222 mg, 0.46 mmol) instead of2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamideand the residue was purified by flash column chromatography (0-15% 2 NNH₃-MeOH/EA) to provide the title compound (97 mg, 69% yield). MS (ESI):mass calcd. for C₁₉H₁₉N₇, 345.17; m/z found, 346.0 [M+H]⁺. ¹H NMR (400MHz, DMSO-d₆): δ 12.61 (s, 1H), 11.86 (s, 1H), 8.55 (s, 1H), 7.92 (d,J=1.3 Hz, 1H), 7.83 (s, 1H), 7.48 (t, J=3.0 Hz, 1H), 6.76 (dd, J=3.5,1.8 Hz, 1H), 5.85 (s, 1H), 2.60 (d, J=6.0 Hz, 2H), 2.57-2.41 (m, 2H),2.14-1.88 (m, 5H), 1.45-1.27 (m, 2H).

Example 3 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide

Step A:2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide.A mixture of ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 3, 555 mg, 1.06 mmol) and cyclopropylmethylamine (1.87 mL,21.1 mmol) was heated at 125° C. for 1 h in a microwave reactor. Theresidue was treated with water then extracted with ethyl acetate. Theorganic layers were combined, dried over sodium sulfate, passed througha silica plug, and concentrated to dryness using a rotovap to providethe title compound (642 mg). MS (ESI): mass calcd. for C₂₈H₃₀N₆O₃S,530.21; m/z found, 531.2 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): δ 8.64 (s,1H), 8.19-8.11 (m, 2H), 7.95 (d, J=4.1 Hz, 1H), 7.66-7.57 (m, 1H),7.57-7.48 (m, 2H), 7.11 (d, J=4.1 Hz, 1H), 4.53 (s, 1H), 4.08 (s, 2H),3.07 (d, J=7.1 Hz, 2H), 2.53 (d, J=5.9 Hz, 2H), 2.45-2.30 (m, 2H),2.14-2.03 (m, 5H), 1.55-1.42 (m, 2H), 1.02-0.94 (m, 1H), 0.54-0.47 (m,2H), 0.24-0.18 (m, 2H).

Step B:2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide.To a mixture of2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfony)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide(560 mg, 1.05 mmol) in 1,4-dioxane (4.22 mL) was added 3N KOH (2.81 mL).The mixture was heated at 80° C. for 1 hr, then purified with basicHPLC: Xbridge Prep OBD Cis 50 mm×100 mm, 5 μm column (eluent 0-100% aqNH₄OH/ACN (10 min)) to provide the title compound (187 mg, 46% yield).MS (ESI): mass calcd. for C₂₂H₂₆N₆O, 390.22; m/z found, 391.2 [M+H]⁺. ¹HNMR (400 MHz, CD₃OD): δ 8.57 (s, 1H), 7.50 (d, J=3.5 Hz, 1H), 6.86 (d,J=3.5 Hz, 1H), 4.57 (s, 1H), 4.11 (s, 2H), 3.13 (d, J=7.0 Hz, 2H),2.67-2.52 (m, 4H), 2.20-2.03 (m, 5H), 1.58-1.44 (m, 2H), 1.12-0.97 (m,1H), 0.60-0.48 (m, 2H), 0.31-0.22 (m, 2H).

Example 4 synthesis and characterization:

N-(2-Cyanoethyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide

To a solution of sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 4, 400 mg, 1.11 mmol) and 3-aminopropanenitrile (320 mg,2.24 mmol) in DMF (5 mL) was added PyBOP (870 mg, 1.67 mmol) and DIPEA(0.60 mL, 3.5 mmol). The reaction mixture was stirred at roomtemperature for 40 h. After removal of the DMF in vacuo, the residue waspurified by flash column chromatography using 50-100% ethyl acetate inheptane. The collected fractions were concentrated in vacuo to a smallvolume and white solid which had precipitated out was filtered off,washed with 10% MeOH in CH₂Cl₂, and dried to provide to provide thetitle compound (75 mg, 17% yield). The filtrate was concentrated todryness and purified by reverse phase-HPLC using a Varian PursuitXR_(s)5 Diphenyl 100 mm×30 mm column (eluent 10-90% CH₃CN in water, 0.1%TFA) to provide a clear oil. This material was dissolved in 10% MeOH inCH₂Cl₂, passed through three 500 mg columns of SILICYCLE SPE-R66030B-03PCarbonate (SiliaBond acid scavenger solid phase extraction cartridge) toremove the TFA and eluted with 10% MeOH in CH₂Cl₂ to provide anadditional fraction of the title compound (88 mg, 20% yield). The twofractions were combined to provide the final product (163 mg, 37% yield)as a white solid. MS (ESI): mass calcd. for C₂₁H₂₃N₇O, 389.20; m/zfound, 390.3 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): δ 8.53 (s, 1H), 7.48 (d,J=3.0 Hz, 1H), 6.84 (d, J=3.5 Hz, 1H), 4.51 (br s, 1H), 4.12 (s, 2H),3.50 (t, J=6.6 Hz, 2H), 2.71 (t, J=6.6 Hz, 2H), 2.45-2.63 (m, 4H),2.03-2.19 (m, 5H), 1.44-1.57 (m, 2H).

Example 5 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)acetamide

A mixture of ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 3, 309 mg, 0.61 mmol) and 4-aminotetrahydropyran (195 mg,1.93 mmol) in 1,4-dioxane (0.5 mL) was heated in a microwave reactor at180° C. for 1 hr. The reaction mixture was then diluted with 1,4-dioxane(1.5 mL), treated with aq 3N KOH (2 mL), and heated at 80° C. for 1.5hr. The residue was then treated with water (10 mL) and extracted withCH₂Cl₂ (3×50 mL). The organic layers were combined, dried over MgSO₄ andconcentrated in vacuo. The crude material was purified using flashcolumn chromatography (5-10% MeOH/CH₂Cl₂) to yield the title compound(146 mg, 57% yield). MS (ESI): mass calcd. for C₂₃H₂₈N₆O₂, 420.23; m/zfound, 421.2 [M+H]⁺. ¹H NMR (600 MHz, DMSO-d₆): δ 11.84 (s, 1H), 8.49(s, 1H), 8.36 (d, J=7.5 Hz, 1H), 7.46 (t, J=3.0 Hz, 1H), 6.73-6.67 (m,1H), 4.54-4.41 (m, 1H), 3.98 (s, 2H), 3.86-3.81 (m, 2H), 3.81-3.74 (m,1H), 3.40-3.34 (m, 2H), 2.60 (d, J=6.0 Hz, 2H), 2.41-2.29 (m, 2H),2.10-2.01 (m, 1H), 2.00-1.93 (m, 4H), 1.77-1.70 (m, 2H), 1.49-1.33 (m,4H).

Example 6 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)acetamide

To a microwave vial were added ethyl2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 3, 300 mg, 0.593 mmol) and 4-aminomethyltetrahydropyran(683 mg, 5.93 mmol). The resulting solution was stirred at 125° C. for 1h. Next were added dioxane (2.37 mL) and KOH (3 M in water, 1.58 mL,4.75 mmol) and the reaction was stirred in the microwave at 80° C. for 1h. The reaction was purified over basic HPLC using a Waters Xbridge PrepOBD Cis 150 mm×30 mm, 5 μm column (eluent 0-100% water (0.05% NH₄OH)/ACN(10 min)) to provide the title compound (97 mg, 38% yield). MS (ESI):mass calcd. for C₂₄H₃₀N₆O₂, 434.24; m/z found, 435.2 [M+H]⁺. ¹H NMR (500MHz, CD₃OD): δ 8.43 (s, 1H), 7.38 (d, J=3.5 Hz, 1H), 6.74 (d, J=3.5 Hz,1H), 4.43 (s, 1H), 4.03-3.96 (m, 2H), 3.89-3.79 (m, 2H), 3.34-3.25 (m,2H), 3.04 (d, J=6.8 Hz, 2H), 2.54-2.38 (m, 4H), 2.08-1.96 (m, 5H),1.74-1.63 (m, 1H), 1.59-1.54 (m, 2H), 1.46-1.33 (m, 2H), 1.25-1.14 (m,2H).

Example 7 synthesis and characterization:

N-(2-Cyano-2-methylpropyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide

A solution of sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 4, 300 mg, 0.835 mmol), 3-amino-2,2-dimethylpropanenitrile(82.0 mg, 0.835 mmol), DIPEA (216 mg, 1.67 mmol), and DMF (5 mL) wasstirred at 0° C. for 1 h. Then PyBrOP (467 mg, 1.00 mmol) was added andthe reaction mixture stirred overnight at room temperature. The mixturewas quenched with 10 mL water and was purified by preparative HPLC usinga Waters Xbridge Prep OBD Cis 150 mm×30 mm 5 μm column (eluent: 28%water (0.05% ammonia hydroxide v/v)-ACN to provide the title compound(58 mg, 16% yield) as a white solid. MS (ESI): mass calcd. forC₂₃H₂₇N₇O, 417.23; m/z found, 418.2 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ11.86 (br s, 1H), 8.71-8.66 (m, 1H), 8.50 (s, 1H), 7.48-7.45 (m, 1H),6.73-6.69 (m, 1H), 4.53-4.42 (m, 1H), 4.10 (s, 2H), 3.33-3.31 (m, 1H),2.57 (d, J=5.6 Hz, 2H), 2.41-2.27 (m, 2H), 2.05-1.93 (m, 5H), 1.45-1.26(m, 9H).

Example 8 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((1-hydroxycyclobutyl)methyl)acetamide

A solution of sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 4, 300 mg, 0.835 mmol), 1-(aminomethyl)cyclobutanol (84.4mg, 0.835 mmol), DIPEA (216 mg, 1.67 mmol), and DMF (5 mL) was stirredat 0° C. for 1 h. Next, PyBrOP (467 mg, 1.00 mmol) was added and wasstirred at room temperature overnight, then quenched with 10 mL water.The reaction was purified by preparative basic HPLC using a Kromasil 150mm×25 mm, 10 μm column (eluent: water (0.05% ammonia hydroxide v/v)-ACNfrom 9% to 39%, v/v) to provide the title compound (90 mg, 26% yield) asa white solid. MS (ESI): mass calcd. for C₂₃H₂₈N₆O₂, 420.23; m/z found,421.2 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 11.58 (br s, 1H), 8.50 (s,1H), 7.94-7.85 (m, 1H), 7.46-7.40 (m, 1H), 6.75-6.70 (m, 1H), 4.89 (brs, 1H), 4.57-4.47 (m, 1H), 4.04 (s, 2H), 3.27 (d, J=6.0 Hz, 2H), 2.55(d, J=6.0 Hz, 2H), 2.45-2.31 (m, 2H), 2.10-1.88 (m, 9H), 1.71-1.60 (m,1H), 1.54-1.35 (m, 3H).

Example 9 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1-methyl-1H-pyrazol-4-yl)acetamide

To a solution of sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 4, 100 mg, 0.278 mmol) and 1-methyl-1H-pyrazol-4-amine(54.0 mg, 0.557 mmol) in DMF (0.8 mL) were added PyBrOP (217 mg, 0.417mmol) and DIPEA (0.144 mL, 0.835 mmol) and the mixture was stirred atroom temperature overnight. The DMF was removed under reduced pressureand the residue was purified by flash column chromatography (50-100%EtOAc/heptanes, then 10% MeOH/DCM) and the subsequently by reverse phaseHPLC using a Varian Pursuit XR_(s)5 Diphenyl 100 mm×30 mm column (eluent10-90% CH₃CN in water, 0.1% TFA) to provide the product as the TFA salt.This material was dissolved in 10% MeOH in CH₂Cl₂ and passed through a500 mg column of SILICYCLE SPE-R66030B-03P Carbonate (SiliaBond acidscavenger solid phase extraction cartridge) to remove the TFA to providethe title compound (34 mg, 29% yield) as a white solid. MS (ESI): masscalcd. for C₂₂H₂₄N₈O, 416.21; m/z found, 417.3 [M+H]⁺. ¹H NMR (400 MHz,CDCl₃) δ=12.77 (br s, 1H), 11.24 (br s, 1H), 7.94 (s, 1H), 7.89 (br s,1H), 7.46 (s, 1H), 7.29-7.20 (m, 1H), 6.74 (br s, 1H), 4.85-4.65 (m,1H), 4.23 (s, 2H), 3.85 (s, 3H), 2.80-2.55 (m, 1H), 2.45 (d, J=6.6 Hz,2H), 2.32-1.98 (m, 6H), 1.62-1.44 (m, 2H).

Example 10 synthesis and characterization:

N-(4-(Cyanomethyl)bicyclo[2.2.1]heptan-1-yl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide

Step A: Dimethyl cyclopentane-1,3-dicarboxylate. A solution ofcyclopentane-1,3-dicarboxylic acid (70.0 g, 443 mmol) and anhydrousmethanol (300 mL) was cooled to 0° C. in an ice water bath. Concentratedsulfuric acid (14 mL) was added dropwise, maintaining the temperature at<15° C. After the addition, the reaction was heated to 90° C. andstirred overnight. The reaction was cooled to room temperature andconcentrated to dryness. The residue was treated with MTBE (500 mL) andH₂O (100 mL). The aqueous layer was separated and extracted with MTBE(2×100 mL). The combined organic extracts were washed with saturatedsodium bicarbonate (2×100 mL), brine (100 mL), dried over anhydrousMgSO₄, filtered, and concentrated to dryness to provide the titlecompound (72.5 g, 88%) as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃) δ3.65 (s, 6H), 2.84-2.72 (m, 2H), 2.26-2.17 (m, 1H), 2.11-2.02 (m, 1H),1.96-1.88 (m, 4H).

Step B: Dimethyl bicyclo[2.2.1]heptane-1,4-dicarboxylate. n-Butyllithium(2.5 M in hexane, 419.0 mL, 1048 mmol) was added slowly to a solution ofdiisopropylamine (152 mL, 1090 mmol) and anhydrous THF (1000 mL) at −78°C. (dry ice/acetone) under N₂. Next, the reaction was stirred for 0.5hours at 0° C. before cooling to −78° C. DMPU (404 mL, 3350 mmol) wasadded via an addition funnel. Then a solution of dimethylcyclopentane-1,3-dicarboxylate (78.0 g, 419 mmol) and anhydrous THF (300mL) was added slowly via an addition funnel. The reaction was warmed to0° C. and stirred for 30 minutes, then cooled to −78° C. and treatedwith a solution of 1-bromo-2-chloroethane (59.0 mL, 712 mmol) andanhydrous THF (200 mL). The reaction was allowed to warm slowly toroom-temperature and was stirred for 12 hours at room-temperature. Thereaction was quenched with saturated aqueous ammonium chloride (400 mL).The reaction was diluted with ethyl acetate (500 mL), the organic layerseparated, and the aqueous layer was further extracted with ethylacetate (2×500 mL). The combined organic extracts were washed with brine(2×300 mL), dried over anhydrous MgSO₄, filtered, and concentrated todryness. The residue was filtered through a pad of silica gel and washedwith ethyl acetate (2000 mL). The filtrate was concentrated to drynessand the residue was purified by flash column chromatography (petroleumether/ethyl acetate, 30:1 to 20:1, gradient elution) to provide thetitle compound (48.5 g, 54%) as white solid. ¹H NMR (400 MHz, CDCl₃): δ3.69 (s, 6H), 2.08-1.99 (m, 4H), 1.91 (s, 2H), 1.73-1.63 (m, 4H).

Step C: 4-(Methoxycarbonyl)bicyclo[2.2.1]heptane-1-carboxylic acid. Amethanol (80 mL) solution of sodium hydroxide (5.145 g, 128.6 mmol) wasadded slowly to a solution of dimethylbicyclo[2.2.1]heptane-1,4-dicarboxylate (27.3 g, 129 mmol) and THF (700mL) at 0° C. and the reaction mixture was stirred at room temperatureovernight. The reaction was concentrated to dryness and the residue wastriturated with MTBE (15 mL). The precipitate was collected byfiltration, washed with MTBE (5 mL), and dissolved in 100 mL of H₂O. Thesolution was acidified to pH=4 with 2 M HCl. The precipitate wascollected by filtration and dried under vacuum to provide the titlecompound (13.0 g, 51.0%) as white solid. The filtrate was extracted withethyl acetate (3×75 mL) and the combined organic extracts were washedwith brine (50 mL), dried over anhydrous MgSO₄, filtered andconcentrated to dryness to provide a second fraction of the titlecompound (8.0 g, 31%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ12.21 (br s, 1H), 3.59 (s, 3H), 1.94-1.86 (m, 4H), 1.74 (s, 2H),1.61-1.54 (m, 4H).

Step D: Methyl4-(((benzyloxy)carbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylate.Diphenylphosphoryl azide (17.1 mL, 78.6 mmol) was added to a solution of4-(methoxycarbonyl)bicyclo[2.2.1]heptane-1-carboxylic acid (13.0 g, 65.6mmol), DIPEA (22.8 mL, 131 mmol), and anhydrous toluene (200 mL) and thereaction mixture was stirred at 110° C. for 2 hours. The reaction wascooled to 50° C. and benzyl alcohol (13.6 mL, 131 mmol) was added andthe reaction mixture was stirred at 110° C. overnight. The reaction wasconcentrated to dryness, dissolved in MTBE (250 mL) and washed with H₂O(150 mL). The organic layer was separated and the aqueous layer wasextracted with MTBE (2×100 mL). The combined organic extracts werewashed with brine (100 mL), dried over anhydrous MgSO₄, filtered, andconcentrated to dryness. The residue was purified by flash columnchromatography (petroleum ether/ethyl acetate, 10:1 to 5:1, gradientelution) to provide an impure product (28.5 g) as pale yellow oil. Theproduct was further purified by preparative acidic HPLC using aPhenomenex Synergi Max-RP 250×50 mm×10 μm column (eluent: 38% to 68%(v/v) CH₃CN and H₂O with 0.1% TFA). The pure fractions were combined andthe volatiles were removed under vacuum. The residue was diluted withH₂O (80 mL), the pH of the solution was adjusted to pH=8 with saturatedaqueous NaHCO₃ solution, and the resulting solution was extracted withCH₂Cl₂ (3×100 mL). The combined organic extracts were washed with brine(75 mL), dried over anhydrous MgSO₄, filtered, and concentrated todryness to provide the title compound (17.3 g, 85%) as a colorless oil.MS (ESI): mass calcd. for C₁₇H₂₁NO₄, 303.2; m/z found, 303.9 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆) δ 7.56 (br s, 1H), 7.37-7.30 (m, 5H), 4.97 (s,2H), 3.58 (s, 3H), 1.90-1.80 (m, 6H), 1.65-1.59 (m, 4H).

Step E: Methyl4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylate. Amixture of methyl4-(((benzyloxy)carbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylate (17 g,56 mmol), di-tert-butyl dicarbonate (18.35 g, 84.06 mmol), MeOH (200 mL)and wet Pd/C (4 g, 10 wt. %, 50% H₂O) was added to a 500 mL round-bottomflask with a hydrogen balloon (13 psi) and was stirred at roomtemperature for 72 hours. The catalyst was filtered off and the filtratewas concentrated to dryness. The residue was purified by flash columnchromatography (petroleum ether/ethyl acetate, 20:1 to 1:1, gradientelution) to provide the title compound (12.0 g, 79.5%) as a white solid.¹H NMR (400 MHz, DMSO-d₆) δ 7.04 (br s, 1H), 3.57 (s, 3H), 1.93-1.78 (m,4H), 1.77 (s, 2H), 1.63-1.53 (m, 4H), 1.35 (s, 9H).

Step F: 4-((tert-Butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylicacid. To a solution of methyl4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylate (5.0g, 19 mmol), THF (40 mL) and MeOH (20 mL) was added aqueous sodiumhydroxide (1.0 M, 46.4 mL, 46.4 mmol) at room temperature and thereaction mixture was stirred at room temperature for 24 hours. Thereaction was concentrated to dryness and the residue was diluted withH₂O (20 mL), acidified to pH=4-5 with 2 M HCl to provide a precipitate.The precipitate was dissolved in 150 mL of ethyl acetate, washed withbrine (45 mL), dried over anhydrous Na₂SO₄, filtered, and concentratedto dryness to provide the title compound (4.74 g, 100% yield) as whitesolid, which was used directly in the next step. ¹H NMR (400 MHz,DMSO-d₆) δ 12.06 (br s, 1H), 7.00 (br s, 1H), 1.87-1.73 (m, 6H),1.58-1.50 (m, 4H), 1.35 (s, 9H).

Step G: tert-Butyl(4-(hydroxymethyl)bicyclo[2.2.1]heptan-1-yl)carbamate. A solution ofborane-tetrahydrofuran complex (1.0 M, 37.1 mL, 37.1 mmol) was addedslowly to a solution of4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylic acid(4.74 g, 18.6 mmol) and anhydrous THF (50 mL) at 0° C. under a nitrogenatmosphere. After the addition was complete, the reaction was stirred atroom temperature overnight. Water (30 mL) was added to the mixtureslowly and it was stirred for additional 30 minutes. The reaction wasconcentrated to dryness and the residue was diluted with ethyl acetate(50 mL), washed with H₂O (15 mL) and brine (10 mL), dried over anhydrousNa₂SO₄, filtered, and concentrated to dryness. The residue was purifiedby flash column chromatography (petroleum ether/ethyl acetate, 2:1) toprovide the title compound (4.0 g, 89% yield) as white solid. TLC(petroleum ether/ethyl acetate, 2:1), R_(f)=0.5. ¹H NMR (400 MHz,DMSO-d₆): 6.88 (br s, 1H), 4.38 (t, J=5.4 Hz, 1H), 3.36 (d, J=5.4 Hz,2H), 1.73 (br s, 2H), 1.64-1.49 (m, 4H), 1.42 (s, 2H), 1.39-1.33 (m,9H), 1.25-1.16 (m, 2H).

Step H: (4-((tert-Butoxycarbonyl)amino)bicyclo[2.2.1]heptan-1-yl)methylmethanesulfonate. Pyridine (2.7 mL, 33 mmol) was added to a solution oftert-butyl (4-(hydroxymethyl)bicyclo[2.2.1]heptan-1-yl)carbamate (2.0 g,8.3 mmol) and anhydrous CH₂Cl₂ (30 mL). The reaction was cooled to 0° C.and methansulfonyl chloride (2.0 mL, 25.0 mmol) was added and themixture was stirred for 3 hours at room temperature. The reaction wasdiluted with CH₂Cl₂ (50 mL) and water (30 mL). The organic layer wasseparated, washed with brine (15 mL), dried over anhydrous MgSO₄,filtered and concentrated to dryness. The residue was purified flashcolumn chromatography (petroleum ether/ethyl acetate, 5:1 to 1:1,gradient elution) to provide the title compound (2.58 g, 97%) as whitesolid. TLC (petroleum ether/ethyl acetate, 1:1), R_(f)=0.85. ¹H NMR (400MHz, CDCl₃) 4.73 (br s, 1H), 4.21 (s, 2H), 2.98 (s, 3H), 1.93-1.90 (m,2H), 1.78-1.62 (m, 6H), 1.53-1.34 (m, 11H).

Step I: tert-Butyl (4-(cyanomethyl)bicyclo[2.2.1]heptan-1-yl)carbamate.To a solution of(4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptan-1-yl)methylmethanesulfonate (2.58 g, 8.07 mmol) and DMSO (25 mL) was added sodiumcyanide (1.20 g, 24.5 mmol). The reaction was heated to 100° C. andstirred for 24 hours. The reaction was diluted with 50 mL of water andextracted with ethyl acetate (3×40 mL). The combined organic extractswere washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered,and concentrated to dryness. The residue was purified by flash columnchromatography (petroleum ether/ethyl acetate, 5:1) to provide the titlecompound (1.8 g, 89% yield) as white solid. ¹H NMR (400 MHz, DMSO-d₆): δ7.00 (br s, 1H), 2.67 (s, 2H), 1.82 (br s, 2H), 1.69-1.52 (m, 6H),1.47-1.40 (m, 2H), 1.37 (s, 9H).

Step J: 2-(4-Aminobicyclo[2.2.1]heptan-1-yl)acetonitrile hydrochloride.To a suspension of tert-butyl(4-(cyanomethyl)bicyclo[2.2.1]heptan-1-yl)carbamate (850 mg, 3.40 mmol)and ethyl acetate (2 mL) at 0° C. was added a solution of HCl in ethylacetate (4.0 M, 10 mL, 40 mmol). After stirring at room temperature for2 hours, the mixture was concentrated under reduced pressure to dryness.The residue was triturated with MTBE (5 mL) and the suspension wasisolated via filtration. The filter cake was washed with MTBE (1 mL) anddried under reduced pressure to afford the title compound (450 mg, 71%)as a white solid. MS (ESI): mass calcd. for C₉H₁₄N₂ 150.12 m/z, found151.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (br.s., 3H), 2.78 (s,2H), 1.90-1.77 (m, 2H), 1.74-1.62 (m, 4H), 1.60 (s, 2H), 1.56-1.46 (m,2H).

Step K:N-(4-(Cyanomethyl)bicyclo[2.2.1]heptan-1-yl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide.To a solution of sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 4, 300 mg, 0.835 mmol),2-(4-aminobicyclo[2.2.1]heptan-1-yl)acetonitrile hydrochloride (138 mg,0.918 mmol), and DIPEA (0.291 mL, 1.67 mmol) in dry DMF (6 mL) was addedPyBrOP (428 mg, 0.918 mmol) at 0° C. The reaction was stirred atroom-temperature for 12 h. The mixture was quenched with 10 mL water andwas extracted with EtOAc (3×20 mL). The combined organic phases werewashed with brine, dried over anhydrous Na₂SO₄, filtered, andconcentrated to dryness. The residue was purified by preparative HPLC(using a Xtimate Cis 150×25 mm×5 μm column (eluent: 23% to 33% (v/v)CH₃CN and H₂O with 10 mM NH₄HCO₃) and by preparative TLC(dichloromethane:methanol=15:1) to provide the title compound (36.6 mg,9% yield) as a white solid. MS (ESI): mass calcd. for C₂₇H₃₁N₇O, 469.3;m/z found, 470.2 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): δ 8.53 (s, 1H), 7.48(d, J=4.0 Hz, 1H), 6.84 (d, J=4.0 Hz, 1H), 4.47 (br s, 1H), 4.07-4.02(m, 2H), 2.63 (s, 2H), 2.61-2.50 (m, 4H), 2.18-1.98 (m, 7H), 1.94-1.84(m, 2H), 1.82 (s, 2H), 1.79-1.68 (m, 2H), 1.62-1.43 (m, 4H).

Example 11 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide

Step A:2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide.The title compound (383 mg, 70%) was prepared in a manner analogous tothat described in Example 1, Step A using 1H-pyrazol-3-amine (465 mg,5.48 mmol) instead of 1-amino-2-methylpropan-2-ol. MS (ESI): mass calcd.for C₂₇H₂₆N₈O₃S, 542.2; m/z found, 543.2 [M+H]⁺. ¹H NMR (500 MHz,DMSO-d₆) δ 12.36 (s, 1H), 11.36 (s, 1H), 10.81 (s, 1H), 8.65 (s, 1H),8.14-8.10 (m, 2H), 7.96 (d, J=4.1 Hz, 1H), 7.72-7.68 (m, 1H), 7.63-7.60(m, 2H), 7.30 (s, 1H), 7.13 (d, J=4.2 Hz, 1H), 6.44-6.41 (m, 1H), 5.41(s, 1H), 4.57 (s, 1H), 4.44 (s, 2H), 4.24 (s, 2H), 2.56 (d, J=6.2 Hz,2H), 2.23-2.13 (m, 2H), 2.02-1.95 (m, 1H), 1.41-1.32 (m, 2H).

Step B:2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide.The title compound was prepared in a manner analogous to Example 1, StepB using2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide(270 mg, 0.500 mmol) instead of2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamideand purified by basic HPLC using a Xbridge Prep OBD Cis 150 mm×30 mm, 5μm, eluent 5% ACN/NH₄OH (aq) (10 min) to provide the title compound (15mg, 7%). MS (ESI): mass calcd. for C₂₁H₂₂N₈O, 402.5; m/z found, 403.2[M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 12.35 (s, 1H), 11.85 (s, 1H), 10.84(s, 1H), 8.50 (s, 1H), 7.58 (d, J=2.3 Hz, 1H), 7.46 (d, J=3.4 Hz, 1H),6.72 (d, J=3.5 Hz, 1H), 6.47-6.39 (m, 1H), 4.64-4.46 (m, 1H), 4.21 (s,2H), 2.57 (d, J=6.1 Hz, 2H), 2.45-2.26 (m, 2H), 2.08-1.88 (m, 5H), 1.39(q, J=11.7 Hz, 2H).

Example 12 synthesis and characterization:

2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-((1-hydroxycyclopropyl)methyl)acetamide

A solution of sodium2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate(Intermediate 4, 300 mg, 0.835 mmol), 1-(aminomethyl)cyclopropanol (72.7mg, 0.835 mmol), DIPEA (216 mg, 1.67 mmol), and DMF (5 mL) was stirredat 0° C. for 1 h. Then PyBrOP (467 mg, 1.00 mmol) was added and stirredat room-temperature overnight. The mixture was quenched with 10 mL waterand was purified by preparative basic HPLC using a Kromasil 150 mm×25mm, 10 μm column (eluent: water (0.05% ammonia hydroxide v/v)-ACN from5% to 35%, v/v) to provide the title compound (84 mg, 25% yield) as awhite solid. MS (ESI): mass calcd. for C₂₂H₂₆N₆O₂, 406.21; m/z found,407.2 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 11.53 (br s, 1H), 8.50 (s,1H), 8.03-7.93 (m, 1H), 7.44-7.40 (m, 1H), 6.74-6.71 (m, 1H), 5.04 (s,1H), 4.58-4.49 (m, 1H), 4.02 (s, 2H), 3.29 (d, J=6.0 Hz, 2H), 2.55 (d,J=6.0 Hz, 2H), 2.45-2.31 (m, 2H), 2.10-1.97 (m, 5H), 1.50-1.37 (m, 2H),0.62-0.55 (m, 2H), 0.55-0.48 (m, 2H).

Polymorph Screening Example

Some embodiments of compounds according to this invention as free basespresent multiple crystalline configurations that have a complexsolid-state behavior, some of which in turn can present distinguishingfeatures among themselves due to different amounts of incorporatedsolvent. Some embodiments of compounds according to this invention arein the form of pseudopolymorphs, which are embodiments of the samecompound that present crystal lattice compositional differences due todifferent amounts of solvent in the crystal lattice itself. In addition,channel solvation can also be present in some crystalline embodiments ofcompounds according to this invention, in which solvent is incorporatedwithin channels or voids that are present in the crystal lattice. Forexample, various crystalline configurations including those given inTable 2, were found for compound Ex. 1. Because of these features,non-stoichiometric solvates were often observed, as illustrated in Table2. Furthermore, the presence of such channels or voids in the crystalstructure of some embodiments according to this invention enables thepresence of water and/or solvent molecules that are held within thecrystal structure with varying degrees of bonding strength.Consequently, changes in the specific ambient conditions can readilylead to some loss or gain of water molecules and/or solvent molecules insome embodiments according to this invention. It is understood that“solvation” (third column in Table 2) for each of the embodiments listedin Table 2 is the formula solvation, and that the actual determinationof the same as a stoichiometry number (fourth column in Table 2) canslightly vary from the formula solvation depending on the actual ambientconditions when it is experimentally determined. For example, if abouthalf of the water molecules in an embodiment may be present ashydrogen-bonded to the active compound in the crystal lattice, whileabout the other half of water molecules may be in channels or voids inthe crystal lattice, then changes in ambient conditions may alter theamount of such loosely contained water molecules in voids or channels,and hence lead to a slight difference between the formula solvation thatis assigned according to, for example, single crystal diffraction, andthe stoichiometry that is determined by, for example, thermogravimetricanalysis coupled with mass spectroscopy. Features of compounds accordingto this invention, such as formula solvation and stoichiometry, aredetermined, once each of such embodiments is made, by techniques such assingle crystal diffraction, and thermogravimetric analysis coupled withmass spectroscopy.

TABLE 2 Some embodiments of crystalline forms of compound Ex. 1 Embodi-Crystallization ment solvent Solvation Stoichiometry  1s — monohydrate0.8 H₂O  1a Water monohydrate 1.3 H₂O  1b Toluene Toluene solvate 0.4toluene  1c Ethyl acetate/ monohydrate 1.1 H₂O 1,4-dioxane  1dAcetonitrile/ 1.7 hydrate 1.7 H₂O chloroform  1e Ethyl acetate/monohydrate 1 H₂O 1,4-dioxane  1f p-xylene p-xylene solvate 0.3 p-xylene 1f Cumene Cumene solvate 0.3 cumene  1g Anisole Anisole solvate 0.3anisole  1h p-xylene p-xylene solvate 0.2 p-xylene  2 1,4-dioxane1,4-dioxane solvate 1.2 1,4-dioxane  3b Cyclohexanone Cyclohexanonesolvate 0.3 Cyclohexanone  3c 1,4-dioxane 1,4-dioxane solvate 0.51,4-dioxane  3d THF THF solvate 0.4 THF  3e Isobutanol Isobutanolsolvate 0.7 isobutanol  1b + 4 Water/ Mix hydrate/ — methanol methanolsolvate  5 Chloroform Chloroform solvate 0.5 chloroform  6 AcetonitrileAnhydrous 0.2 acetonitrile  1s + 7 Heptane Heptane solvate 0.1 heptane 7 — Non-solvated —  8 — Non-solvated —  9 — Non-solvated — 10 dihydrate1.8 H₂O 11 ethanol ethanol solvate 0.5 ethanol 11b methanol methanolsolvate 0.5 methanol 12 — anhydrous — 13 methanol/water metastable form14 metastable hydrate 15 toluene toluene solvate 0.55 toluene 16 ethylacetate ethyl acetate 0.09 ethyl solvate acetate 17 isopropyl isopropyl0.13 isopropyl acetate acetate solvate acetate 18 2-butanone 2-butanonesolvate 0.2 2-butanone

The compound that was obtained as described in Example 1 was furthercrystallized by preparing a slurry in DCM (1:3, for example 10 g ofcompound in 30 ml DCM) that was stirred at 40° C. for 4 hours, andfurther stirred for 14 hours at 25° C., then heptane was slowly added(1:2, for example 20 ml of heptane into the compound/DCMslurry/solution) at 25° C., stirred at 40° C. for 4 hours, cooled to 25°C. and stirred for further 14 hours at 25° C. Subsequent filtration leadto compound Ex. 1 in the form of an off-white solid, that was identifiedas a monohydrate, a 1 s embodiment.

An amorphous form of compound Ex. 1, embodiment 19, was prepared asfollows. Embodiment is (1 g) was dissolved in t-butanol (40 vol) andstirred at 50° C. Pre-dried molecular sieves were added to the solutionand stirred for 10 min. The solution was filtered and aliquoted intoHPLC vials (1 ml) which were frozen in a dry ice-acetone bath rightafterwards. Samples were then placed on the freeze drier for 48 h. Thematerial was amorphous by XRPD and consistent with the proposedstructure by ¹H-NMR, with 0.4 mol of t-butanol per molecule of compoundEx. 1 present. This material was heated to 150° C. and held at 150° C.for 10 min. The final sample was analyzed by XRPD and ¹H-NMR and it wasdetermined to be amorphous and that 0.03 mol of t-butanol remained permolecule of compound Ex. 1.

Embodiments 1-18 in Table 2 and FIG. 2 are crystalline, and embodiment19 in FIG. 2 is amorphous. Embodiments 1s and 1a through 1h areisostructural. Embodiment 1s crystallizes in a centro-symmetricaltriclinic space group P-1. The term “embodiment 1” collectively refersto the isostructural embodiments 1s and 1a through 1h. Any one of such1s and 1a through 1h embodiments is sometimes referred to as anisostructural member of embodiment 1 or just as a member ofembodiment 1. Embodiments 3b, 3c, 3d and 3e are isostructural andcrystallize in the monoclinic system, space group C 2/c. The term“embodiment 3” collectively refers to the isostructural embodiments 3b,3c, 3d and 3e. Any one of such 3b, 3c, 3d and 3e embodiments issometimes referred to as an isostructural member of embodiment 3 or justas a member of embodiment 3. Isostructural embodiments are such thatthey possess similar crystal structure properties (same symmetries andsimilar unit cell parameters and crystal packing) while having differentchemical compositions (i.e., different solvent and/or water moleculesincorporated in the crystal lattice). Unit cell parameters inisostructural embodiments can slightly differ due to the differentcomposition (solvent or water incorporated into the crystal structure).Embodiments referred to in Table 2 were prepared and/or inter-convertedas schematically shown in FIG. 2 and as described in more detail asfollows.

Crystallization protocols used in these preparations included solventequilibration in neat solvents, evaporative crystallization, coolingcrystallization with hot filtration, crash-crystallization withanti-solvent, crystallization by thermocycling, incubation at lowtemperature, heat/cool maturation, incubation at elevated temperature,high temperature maturation using amorphous material (embodiment 19),and thermocycling using amorphous material (embodiment 19). Solids wereanalyzed by HT-XRPD or XRPD. When applicable, mother liquors wereevaporated completely and the remaining solids were also analyzed byHT-XRPD or XRPD. The starting material embodiment 1s as a monohydratewas a predominant solid form.

Solvent equilibration at 25° C. and 50° C.

Long term slurry experiments were performed by suspending compoundembodiment 1s in twenty neat solvents and stirring at room temperaturefor two weeks and at 50° C. for one week. Upon completion of theequilibration time, the residual solids were separated from the motherliquors. The solids were dried under ambient conditions and dried undervacuum (5 mBar) before being analyzed by HT-XRPD. Subsequently, thesolids were exposed to accelerated aging conditions (40° C./70% relativehumidity) for two days and again analyzed by HT-XRPD.

From most of the crystallization solvents, the starting material asembodiment 1s was obtained. From several crystallization solvents,HT-XRPD patterns were found to be similar to those of the initialembodiment 1s. In most of these diffraction patterns, peak shifts and/oradditional peaks were identified. Each of these patterns corresponded toan embodiment that was labeled as one of 1a through 1h, and based on thesimilarities in the HT-XRPD diffraction patterns for such embodiments,they are presented as embodiments that are isostructural members ofembodiment 1. All isostructural members of embodiment 1 converted toembodiment 1a after exposure to 40° C. and 75% RH for two days.

Embodiment 1s converted to hydrated embodiment 10 when it was exposed to100% RH at 25° C. Nevertheless, embodiment 10 was physically not stableat ambient conditions. Whereas embodiment 1s crystallized in thetriclinic system, space group P-1, embodiment 10 was found tocrystallize in the monoclinic system, space group C 2/c. Embodiment 10had limited physical stability under ambient conditions and it convertedto another embodiment such as 1s or 1a. This behavior is attributable toan unequally strong binding of all the hydration/solvation molecules. Inthis case, embodiment 10 would have a less strongly bound second watermolecule that would be lost under ambient conditions. More precisely,the physical stability of embodiment 1s was investigated in climatechambers by exposing a 20 mg sample of such embodiment to 40° C. and 70%relative humidity for four days, and another 20 mg sample of the sameembodiment was exposed also for four days to 25° C. and 100% relativehumidity. After four days, the various solid samples were analyzed byHR-XRPD, the crystal cell parameters were determined and thediffractograms were indexed. Diffractograms are shown in FIG. 6. Frombottom to top, the first diffractogram in FIG. 6 corresponds toembodiment 1s as starting material, and the second corresponds to thesame form after a 4-day exposure to 40° C. and 70% relative humidity,noted as “1s 70 RH” in the same figure. This analysis revealed that theinitial embodiment 1s had been recovered although with a small amount ofa second crystalline form that was possibly another hydrated embodimentwith a higher water content. Indexing for such form was not possible dueto the small amount in which it was present. The third diffractogramcorresponds to embodiment 1s after a 4-day exposure to 25° C. and 100%relative humidity, noted as “10” in the same figure. These conditionslead to the conversion of embodiment 1s into embodiment 10, with a smallcontamination of initial embodiment 1s, and solvation as characterizedin Table 2. Upon dehydration, both embodiments 1s and 10 re-crystallizedto the anhydrous form with a melting point of 148° C.

Embodiment 10 was also prepared by slurry conversion of embodiment 1s orembodiment 19 in water, with temperature cycling of 25-5° C. The slurrywas prepared by suspending 50 mg of material in 1 mL water. Temperaturecycling of 25-5° C. is: the mixture was heated at 25° C. for 1 hour andthen the temperature was decreased to 5° C. over a 2 hour period. Themixture was then held at a temperature of 5° C. for 1 hour. Thetemperature was then increased to 25° C. over a 2 hour period. Thistemperature cycling regime was repeated for a total of about 24 hours.The solids were isolated by vacuum filtration and then dried on a filterfor 10 minutes. Embodiment 10 converted to embodiment 1s during dryingunder ambient conditions and under vacuum.

Solvent equilibration at room temperature yielded embodiment 1b out oftoluene as the crystallization solvent, and embodiment 1f out ofp-xylene as the crystallization solvent.

Three additional solid embodiments were identified and designated asembodiments 2, 3 and 7. Embodiment 2, whose TGA and DSC are shown inFIGS. 21A and 21B, respectively, was identified from the solventequilibration experiment performed at room temperature in 1,4-dioxanewhile embodiment 7 was found as a mixture with embodiment 1s in thesingle solvent equilibration experiment at 50° C. from heptane. Severalsimilar but not identical diffractograms were identified which weregrouped as embodiments 3b, 3c, 3d and 3e that are isostructural membersof embodiment 3. Isostructural members of embodiment 3 were found mixedwith members of embodiment 1. The mixtures containing members ofembodiment 3 transformed in some cases to embodiment 1a or to mixturesof embodiments 1a and 3e. Embodiment 7 appeared to be physically stable,but embodiment 2 converted to embodiment 3e after exposure to AAC fortwo days.

Evaporative Crystallization

The mother liquors saved from the solvent equilibration experimentsperformed at RT were used for slow evaporative crystallizationexperiments. The mother liquors were filtered to remove any particulatematter and allowed to slowly evaporate under ambient conditions. Theobtained solids were analyzed by HT-XRPD and again after exposure to AACfor two days.

Due to the poor solubility of compound Ex. 1 in some of the solvents, nosolids were recovered when such solvents were used. In the experimentswhere solids had precipitated, an amorphous residue or isostructuralmembers of embodiments 1 or 3 were recovered. During the stabilitystudy, the different members of embodiment 1 converted to embodiment 1awhilst the sample of embodiment 3 seemed to be physically stable. Theamorphous solids in some cases remained amorphous after the stabilitystudy, became deliquescent or showed some signs of crystallinity.

Cooling Crystallization

The mother liquors of the solvent equilibration experiments performed at50° C. were filtered at 50° C. to remove any particulate matter. Thesuspensions at 50° C. were filtered using 0.2 m PTFE filters, and thesolutions were placed at 5° C. and aged for 72 hours. When solids hadprecipitated during aging these solids were separated from the liquid,dried under ambient conditions and under vacuum, and analyzed byHT-XRPD. The remaining mother liquors were allowed to slowly evaporateand the remaining solids were analyzed by HT-XRPD. The samples in whichno precipitation occurred were placed under vacuum and the dried solidswere analyzed by HT-XRPD. All the solids were then exposed to AAC (2days at 40° C./70% RH) and re-analyzed by HT-XRPD.

Solids did not precipitate upon cooling in some of the solutions, inwhich cases the solutions were evaporated under ambient conditions. Dueto the low solubility of compound Ex. 1 in some solvents, no solids wereobtained from some solutions.

From four solvents (2-propanol, 2-butanone, acetonitrile, and methanol),precipitation occurred. Embodiment 6 was identified after evaporation ofa single cooling crystallization experiment at mL scale in 800 μLacetonitrile, concentration of 25 mg/mL. Embodiment 6 seemed to be astable solid form after 2 days AAC, and it appeared as a non-solvatedembodiment.

Cooling/Evaporative Crystallization at μL Scale

The cooling/evaporative crystallization experiments at μL scale wereperformed in a 96-well plate, using 12 neat solvents and 12 solventmixtures and applying four temperature profiles. In each wellapproximately 4 mg of embodiment 1s was solid dosed. Subsequently, thecrystallization solvents (80 μL) and solvent mixtures were added toreach a concentration of 50 mg/mL, and the plate, with each wellindividually sealed, to subsequently undergo one of the four temperatureprofiles. Upon completion of the temperature profile the solvents wereallowed to evaporate at low ambient pressure (24 hours) and theremaining solids were analyzed by HT-XRPD before and after exposure toAAC for 2 days (40° C./70% RH).

Members of embodiments 1 and 3 were found from most of the solventsystems and temperature profiles. However, a certain tendency of solidform versus temperature profile was observed. Embodiment 1b was mainlyidentified from the short temperature profiles (3 hours aging).Nevertheless, the same solvent systems with long aging times led to theidentification of embodiment 1f, members of embodiment 3 or mixtures ofmembers of embodiments 1 and 3. Embodiment 3c was obtained with1,4-dioxane as crystallization solvent and a temperature profile of 50°C. as initial temperature, held for 60 min, followed by cooling at arate of 1° C./h to a final temperature of 20° C., held for 48 h;embodiment 3d was obtained with tetrahydrofuran as crystallizationsolvent and the same temperature profile as for embodiment 3c.

Embodiment 4 was identified in experiments performed in methanol/water(50/50, v/v), THF and DCM/IPA (50/50, v/v) when short aging conditionswere applied. Embodiment 4 was obtained by treating embodiment 1s with amixture (50/50) of water and methanol and a temperature profile of 50°C. as initial temperature, held for 60 min, followed by cooling at arate of 20° C./h to a final temperature of 5° C., held for 3 h, whichyielded embodiment 4 together with embodiment 1b. Embodiment 4 togetherwith embodiment 1b was also obtained by treating 1s with a mixture(50/50) of water and methanol and a temperature profile of 50° C. asinitial temperature, held for 60 min, followed by cooling at a rate of20° C./h to a final temperature of 20° C., held for 3 h. Embodiment 4did not appear to be physically stable under ambient conditions. Coolingcrystallization experiments yielded embodiment 1c out of ethylacetate/1,4-dioxane (50/50, v/v) as the crystallization solvent and atemperature profile of 50° C. as initial temperature, held for 60 min,followed by cooling at a rate of 1° C./h to a final temperature of 5°C., held for 48 h; embodiment 1d out of acetonitrile/chloroform (50/50,v/v) as the crystallization solvent and a temperature profile of 50° C.as initial temperature, held for 60 min, followed by cooling at a rateof 1° C./h to a final temperature of 5° C., held for 48 h; andembodiment 1e out of ethyl acetate/1,4-dioxane (50/50, v/v) as thecrystallization solvent and a temperature profile of 50° C. as initialtemperature, held for 60 min, followed by cooling at a rate of 1° C./hto a final temperature of 20° C., held for 48 h.

Embodiment 5 was identified in experiments performed in chloroform asthe crystallization solvent and a temperature profile of 50° C. asinitial temperature, held for 60 min, followed by cooling at a rate of1° C./h to a final temperature of 20° C., held for 48 h.

Similar conversions were seen during the stability study as previouslyobserved in the other crystallization methods. In most cases all solidforms converted to embodiment 1a or to mixtures containing embodiment1a.

Evaporative Crystallization from Solid Mixtures

In evaporative crystallization using solvent/anti-solvent mixtures,clear solutions of a compound are prepared from which the solventevaporates first (high vapor pressure) causing the compound toprecipitate to some extent in the form of crystals. These crystals thenact as seeds when the anti-solvent (lower vapor pressure) is evaporated.

Compound Ex. 1 did not completely dissolve in each of the solventsystems. For that reason, all the experiments included filtration priorto evaporation.

The results of the HT-XRPD analysis demonstrated that compound Ex. 1crystallized mainly as embodiment 1s upon evaporation of solventmixtures. This was observed for the following solvent/anti-solventsystems: tetrahydrofuran/water, acetonitrile/water, chloroform/ethanol,methanol/ethyl acetate, 2-butanone/isopropanol, and heptane/acetone.From two systems, acetone/cumene and 1,4-dioxane/ethyl formate, theisostructural embodiments 3b and 3e were identified, which after AACconverted to different mixtures of embodiments 1a and 3d, and 1s and 3e,respectively.

Anti-Solvent Crystallization

Saturated solutions of compound Ex. 1 were prepared in neat solvents.The anti-solvent additions were performed in forward and reverseadditions. In the forward addition, the anti-solvent was added in threealiquots to the compound solution. The reverse addition was performed byadding a volume of compound solution to a large excess of anti-solvent(20 mL).

After precipitation, the solids were separated from the liquids, driedunder ambient conditions and dried under vacuum (5 mbar) before beinganalyzed by HT-XRPD. The experiments in which no precipitation occurredupon anti-solvent addition were stored at 5° C. for 48 hours to induceprecipitation. The precipitated solids were afterwards separated andanalyzed by HT-XRPD. When no solids were obtained, the solutions wereevaporated under mild conditions and the residual solids were analyzedby HT-XRPD. All solids were exposed to AAC (2 days at 40° C./70% RH) andwere re-analyzed by HT-XRPD.

The forward anti-solvent crystallization showed precipitation in allcases. All solids could be classified as isostructural members (1s, 1b,1j, 1f) of embodiment 1 or of embodiment 3 (3b, 3d, 3f). After exposureto AAC, all solid samples converted to embodiment 1a, except one thatconverted to a mixture of embodiments 1a and 3e.

The reverse anti-solvent crystallization experiments performed in DMSOas solvent gave different solid forms depending on the anti-solventused. With dichloromethane or p-xylene isostructural members (1s and 1b)of embodiment 1 were identified, while with MTBE an amorphous residuewas obtained. Evaporation of two solutions with heptane and water asanti-solvents that had no precipitated upon anti-solvent addition led toan oil. Conversions to embodiment 1a were observed after AAC, and theamorphous residues became deliquescent.

Hot Filtration Experiments

The cooling crystallization experiments with hot filtration wereperformed from supersaturated solutions of compound Ex. 1 prepared at50° C. in different solvent mixtures. The hot filtrated solutionsunderwent a 48-hour cooling profile. The vials in which solids hadprecipitated after the temperature profile were centrifuged and thesolids were separated from the liquid and analyzed by HT-XRPD (afterdrying under vacuum). If no solids had precipitated the solutions wereevaporated under vacuum and the solids analyzed by HT-XRPD. All thesolids were exposed to AAC (2 days at 40° C./70% RH) and re-analyzed byHT-XRPD. In half of the hot filtration experiments precipitation did notoccur and upon evaporation of the solvents, not enough solids wererecovered due to the poor solubility of compound Ex. 1 in those solventsystems. In three experiments, an amorphous residue was recovered whichafter AAC crystallized to a mixture of members of embodiment 1 (1s or1a) and 3 (3e) or became deliquescent. Embodiment 5 was identified fromthe experiment in acetone/chloroform (50/50, v/v). This embodimentappeared to be physically unstable as conversion to embodiment 1a wasobserved after AAC.

Thermo-Cycling Experiments

Suspensions of about 6 mg of embodiment 1s were prepared in 10 solventsat room temperature. The suspensions were cycled between 5° C. and 50°C. Upon completion of the thermo-cycling, the solids were separated bycentrifugation and dried under ambient conditions and under vacuum (5mbar) before being analyzed by HT-XRPD. Subsequently, all solids wereexposed to AAC for two days and again analyzed by HT-XRPD.Thermo-cycling experiments usually promote the formation of the morestable polymorphic form. With the exception of the experiment performedin cyclohexanone all vials contained solids after the thermo profile.The cyclohexanone solution was slowly evaporated under mild vacuum.Members of embodiments 1, 3 or mixtures of them were identified mainlyin the wet solids. Upon drying these solids, conversion to embodiment 1swas observed. Embodiments 3b and 3e were obtained from thermo-cycling in300 μL of cyclohexanone at a concentration of 51 mg/mL (3b), and in 400μL of isobutanol at a concentration of 37.3 mg/mL (3e). Embodiment 5 wasobtained from thermo-cycling in 800 μL of chloroform at a concentrationof 18.6 mg/mL.

FIGS. 3, 4 and 5 show an overlay of HT-XRPD patterns for some of theembodiments listed in Table 2 and also referred to in the screeningsdescribed above.

Embodiment 1s was recovered from most of the crystallizationexperiments. It is a channel hydrate having a variable number of watermolecules and/or other solvents incorporated depending on ambientconditions. Conversion to embodiment 1a was observed. This formcontained slightly more water (1.3 molecules of water). Allisostructural members of embodiment 1 converted to embodiment 1a afterexposure to 40° C. and 75% RH for two days. The shifts of somediffraction peaks in XRPD patterns for members of embodiment 1 might beattributed to the different solvent or water molecules that wereincorporated into the crystal lattice. FIG. 4 shows an overlay ofHT-XRPD patterns for members of embodiment 1. Diffractogram 1scorresponds to compound Ex. 1 as starting material in the form ofembodiment 1s. Diffractogram 1a corresponds to embodiment 1a that wasobtained after exposure to AAC of several embodiment 1s samples.Diffractogram 1b corresponds to embodiment 1b that was obtained from thesolvent equilibration experiment at RT in toluene. Diffractogram 1ccorresponds to embodiment 1c that was obtained from the coolingcrystallization experiment at μL scale in ethyl acetate/1,4-dioxane(50/50, v/v). Diffractogram 1c corresponds to embodiment 1d that wasobtained from the cooling crystallization experiment at μL scale inacetonitrile/chloroform (50/50, v/v). Diffractogram 1e corresponds toembodiment 1e that was obtained from the cooling crystallizationexperiment at μL scale in ethyl acetate/1,4-dioxane (50/50, v/v).Diffractogram if corresponds to embodiment 1f that was obtained from thesolvent equilibration experiment at RT in p-xylene. Diffractogram 1gcorresponds to embodiment 1g that was obtained from the solventequilibration experiment at 50° C. in anisole. Diffractogram 1hcorresponds to embodiment 1h obtained from the cooling crystallizationexperiment at μL scale in p-xylene.

Diffractograms for members of embodiment 3 are shown in FIG. 5. Theshifts observed in the different HT-XRPD patterns are most likelyattributed to the different solvent molecules that were incorporatedinto the crystal lattice. Embodiment 3 was obtained by heatingembodiment 2 to 40° C. at 70% RH for 4 days. Embodiments 3b through 3ewere solvated forms containing a non-stoichiometric amount of solventwhich varied depending on the solvent incorporated in the crystalstructure (0.3-0.7 molecules). The mixtures containing members ofembodiment 3 were unstable upon exposure to AAC and they transformed insome cases to embodiment 1a or to mixtures of embodiments 1a and 3e.Conversion to embodiment 1a is attributed to the exchange of solventmolecules by water molecules upon exposure to high relative humidity,and re-crystallization to the hydrated embodiment 1a.

Embodiment 9 was obtained by heating embodiment 2 to a temperature ofabout 200° C. followed by cooling to 25° C. and also by cyclic DSC25-200-25-300° C. Embodiment 9 was also obtained by additionalprocedures. One of such procedures was a two-step procedure: Embodiment1s (1.5 g) was treated with 1,4-dioxane (10 vol) at RT. Seeds ofembodiment 2 (5 mg) were added and the sample was stirred at RT for 24hours. The resulting suspension was filtered and the sample wasair-dried for 1.5 hours. This sample was determined to be embodiment 2by XRPD. In the second step of this two-step procedure, embodiment 2 washeated to 210° C. at 10° C./min and held at 210° C. for 30 min. Thesample was then allowed to cool to RT. The resulting solid wasdetermined to be embodiment 9 by XRPD analysis. Another of suchprocedures was also a two-step procedure for obtaining embodiment 9. Inthis procedure, embodiment 1s (1.5 g) was treated with 1,4-dioaxne (10vol). Seeds of embodiment 2 (5 mg) were added and the sample was stirredat RT for 24 hours. The resulting suspension was filtered and the samplewas air-dried for 1.5 hours. This sample was determined to be embodiment2 by XRPD. In the second step of this procedure, embodiment 2 was heatedto 150° C. at 10° C./min followed by further heating to 170° C. at 2°C./min. The sample was then allowed to cool to RT. The resulting solidwas determined to be embodiment 9 by XRPD analysis. The TGA and DSC ofembodiment 9 is shown in FIGS. 22A and 22B, respectively.

Embodiment 1s was obtained by slurring embodiment 9 in the followingsolvents for 6 days at 50° C.: 2-butanone, acetone/water (90/10, v/v)and acetonitrile/water (90/10, v/v). Embodiment 1s was also obtainedwhen the same experiment was performed at room temperature.

Embodiment 8 was obtained by heating embodiment 5 to a temperature ofabout 175° C. Embodiment 8 was also obtained by additional procedures.One of such procedures was a two-step procedure: Embodiment 1s (1.5 g)was treated with 1,4-dioxane 10 (vol) and stirred at RT for 72 hours.The resulting suspension was filtered and the solid that was obtainedwas dried in a vacuum oven at RT for 16 hours. The solid obtained fromthis first step was determined by XRPD to be embodiment 3c. In thesecond step, embodiment 3c (100 mg) was heated to 150° C. at 10° C./min,then heated at the slower rate of 2° C./min up to 180° C. The sample wasthen allowed to cool back to RT. The resulting solid was determined byXRPD to be embodiment 8. Another of such procedures was also a two stepprocedure for obtaining embodiment 8. In this procedure, embodiment 19(300 mg) was treated with 1,4-dioxane (3 vol) and shaken at 60° C. for24 hours. The resulting suspension was filtered and the solid obtainedfrom this first step was determined by XRPD to be embodiment 3c. In thesecond step, embodiment 3c (300 mg) was heated to 180° C. at 10° C./min.The sample was then allowed to cool back to RT. The resulting solid wasdetermined by XRPD to be embodiment 8. The TGA and DSC for embodiment 8is shown in FIGS. 20A and 20B, respectively.

In addition to the preparation of embodiment 6 as described above, thisembodiment was obtained by heating embodiment 11 (80-100 mg), whosepreparation is described below, by thermal gravimetric analysis fromambient to 185° C. at 10° C./min and was held isothermally for 3minutes. The sample was then allowed to cool to RT. Embodiment 6 wasalso obtained from embodiment 11 by subjecting it to a slurryexperiment. The slurry experiment was run as follows: the solvent wasadded to embodiment 11 (50 mg) and the mixture was stirred at thedesignated temperature for 0.5 hours. Seed crystals of form 9 (5 mg)were added and the mixture was stirred overnight at the designatedtemperature. The solids were isolated by centrifugation and analyzed byXRPD. using isopropyl acetate (0.5 mL) at both 30° C. and 50° C. Thegeneration of embodiment 6 was confirmed by XRPD. The TGA and DSC forembodiment 6 is shown in FIGS. 19A and 19B, respectively.

Additional embodiments of the invention were obtained as describedbelow.

Embodiment 5 was converted to embodiment 9 by subjecting it to slurryexperiments Slurry experiments were conducted as follows using varioussolvents at the temperatures identified: The solvent was added toembodiment 5 (50 mg) and the mixture was stirred at the designatedtemperature for 0.5 hours. Seed crystals of form 9 (5 mg) were added andthe mixture was stirred overnight at the designated temperature. Thesolids were isolated by centrifugation and analyzed by XRPD. Slurryexperiments run at 50° C. were conducted using the following solvents:TBME (0.75 mL) and a 33:67 mixture of isopropyl acetate: heptane (0.5mL). Slurry experiments run at 75° C. were conducted using the followingsolvents: isopropyl acetate (0.5 mL) and methyl ethyl ketone (0.5 mL).

Embodiment 11 was obtained as follows: A suspension of embodiment 1s (45g) in ethanol (absolute, water content <0.1%, 300 mL) at 50° C. wasstirred for 16.5 hours. The suspension was then cooled to 5° C. at 0.25°C./minute. Subsequently, the suspension was stirred at 5° C. for 3hours. The solids were then filtered off and washed with cold (5° C.)ethanol (absolute, water content <0.1%, 90 mL), and dried under vacuumat 40° C. for 17 hours to yield approximately 39 g of embodiment 11. TheTGA and DSC of embodiment 11 is shown in FIGS. 17A and 17B,respectively.

Embodiment 11 was also obtained as follows: Absolute ethanol (170 mL)was added to embodiment 1s (19 g) and heated to about the boiling pointof the solvent. A small amount of the solids (5%) did not dissolve andwere removed by hot filtration. It was determined that the solids thatwere filtered off, were embodiment 1s. So the solids were added backinto the filtrate and this mixture was heated until all the solidsdissolved. To this hot solution was added, heptane (535 mL), drop-wisevia a separatory funnel. During this drop-wise addition of heptane, thehot solution was stirred vigorously. After the addition of heptane wascomplete, the flask containing the hot solution/heptane mixture wassubmerged in an ice water bath and vigorously stirred for one hour. Thesolids were then collected by filtration and the white solid filter cakewas dried by pulling air through it for 15 minutes. It was further driedby heating it at 70° C. for 16 hours under high vacuum and then byheating it at 80° C. for 18 hours to yield 16.3 g of embodiment 11. Thediffractogram for embodiment 11 is shown in FIG. 7.

In a hygroscopicity study of embodiment 11, it was found it to be onlyslightly hygroscopic, with a mass change of 0.66% between 0-90% RH inthe GVS analysis as shown in FIG. 18. XRPD analysis post GVS analysisshowed that the material was physically stable. Variable temperature(VT)-XRPD was performed in order to assess the stability of embodiment11 upon heating. The material remained unchanged as shown by XRPDanalysis when it was subjected to temperatures up to ca. 175° C.,however above 180° C. the sample converted to embodiment 6. Thediffractograms of embodiment 11 before and after the VT-XRPD experiment,along with the diffractogram for embodiment 6 are shown in FIG. 24.Embodiment 11 was also subjected to static storage analysis at 40°C./75% RH for up to 48 days. The samples were analyzed by XRPD and KarlFisher (KF) after 2 days, 5 days and 48 days. Embodiment 11 remainedunchanged as shown by XRPD analysis with a total water uptake of 1.2%after 48 days. ¹H-NMR of the material post 48 days static storage showedthe material retained 0.36 mol eq of ethanol. Embodiment 11 stored underambient conditions for the period of the study was shown to contain 0.46mol eq of ethanol by ¹H-NMR.

Embodiment 11b was obtained from embodiment 1s as follows: 10 mL ofdried methanol was added to 3.3 g of embodiment 1s. This mixture wassubjected to the following temperature cycling: The mixture was heatedat 40° C. for 1 hour and then the temperature was increased to 60° C.over a 2 hour period. The mixture was then heated at 60° C. for 1 hour.The temperature was then decreased to 40° C. over a 2 hour period. Thistemperature cycling regime was repeated for a total of about 20 hours.At that time the mixture was cooled to 5° C. over a 2 hour period. Thesolids were isolated at 5° C. by vacuum filtration and then dried atambient temperature under vacuum for about 66 hours. Alternatively,embodiment 11b was obtained from embodiment 1s using the followingprocedure: 1 mL of dried methanol was added to 330 mg of embodiment 1s.This mixture was subjected to the following temperature cycling: Themixture was heated at 40° C. for 1 hour and then the temperature wasincreased to 60° C. over a 2 hour period. The mixture was then heated at60° C. for 1 hour. The temperature was then decreased to 40° C. over a 2hour period. This temperature cycling regime was repeated for a total ofabout 18 hours. At that time the solids were isolated by centrifugationand then dried at ambient temperature under vacuum for about 33 hours.The methanol for the above experiments was dried using molecular sieves(3 Å, activated at 100° C. under vacuum for at least 24 h). Thediffractogram for embodiment 11b is shown in FIG. 7E.

Embodiment 12 was obtained from embodiment 1s, which was exposed tohumidity conditions below 10% RH at 25° C. to provide embodiment 12. Thediffractogram for embodiment 12 is shown in FIG. 7B.

Embodiment 13 was obtained as follows: To a 250 mL 4-necked flask at25±5° C. was added a sample of embodiment 1s. The flask was then chargedwith MeOH (4.0 V, 40 mL) and purified water (10 mL, 1.0 V) and stirreduntil all the solid dissolved. N₂ was bubbled into the mixture for 1hour and the mixture was then cooled to 0 to 5° C. A 0.225 mL volume ofa cooled solution (0 to 5° C.) of NaBH₄/water (0.006 eq., 2.5% w/w) wasprepared with purified water (40 mL) charged into a 100 mL of a 4-neckedflask under N₂ at 0° C., followed by the addition of NaBH₄ (1.0 g); themixture was stirred at 0° C. until all the NaBH₄ dissolved. Such NaBH₄solution was added into the 250-mL flask that was cooled (0 to 5° C.)and stirred at 0 to 5° C. The color of the reaction mixture changed toyellow. Purified water (40 mL, 4.0 V, degassed with N₂ before using) wasadded dropwise over 1 hour at 0 to 5° C. The reaction was stirred for 4hours under N₂ at 0 to 5° C. Additional purified water (30 mL, 3.0 V,degassed) was added dropwise over 1 hour at 0 to 5° C. and the reactionmixture was stirred for an additional 16 hours under N₂ at 0 to 5° C.The reaction was then filtered and the resulting solids were washed withpurified water (20 mL, 2 V, degassed with N₂ before using) in a glovebox environment under N₂ (02 content being 200 ppm). The solids weredried under vacuum with moisturized nitrogen at 35±5° C. to provideembodiment 13 as an off-white solid. The diffractogram for embodiment 13is shown in FIG. 7C.

Embodiment 14 was prepared as follows:2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide(48.15 kg, prepared in Ex. 2, Step B), EtOH (technical grade, 481 L) andKOH (6.613 kg) were stirred at 10-20° C. for 9 hours. The reaction wasthen quenched with acetic acid (6.74 L) maintaining the temperature at10-20° C. Acetonitrile (240 L) was added and the solvents wereevaporated under reduced pressure to a volume of about 240 L. Thisaddition and evaporation of acetonitrile was repeated two more times.The resulting mixture was heated to 60-70° C. for 5 hours after which itwas cooled to 10-15° C. and stirred for 2 h. The solids in this mixturewere then filtered off and washed with acetonitrile (48 L) twice. Thesolids were then added to water (240 L) and the reaction mixture heatedto 45-50° C. for 3-5 hours followed by cooling to 15-20° C. for 4 hours.The solids remaining were filtered off and the filter cake was washedwith water (96 L, two times). This filter cake was dried at 45° C. toprovide embodiment 14 (26.28 kg). The diffractogram for embodiment 14 isshown in FIG. 7D.

Additional embodiments of the invention were obtained as describedbelow.

Solubility Assessment

Embodiment 1s (15 mg) was treated with increasing volume of solventuntil the material fully dissolved or until a maximum of 100 mL ofsolvent had been added. The solvent was added in the followingincrements: 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 70 mL and 100 mL.After each addition of solvent, the system was held at 50° C. for 5 minwith gentle stirring and visually assessed for presence of solid. Thisprocess continued until a total of 100 mL of solvent had been added. Ifno solid remained, then no additional solvent was added. After theassessment was completed, the solution was held at 50° C. for 1 h andthen cooled from 50° C. to 5° C. at 0.1° C./min with stirring. If solidwas present, then the mixture was filtered under vacuum using a 96 wellplate and analyzed by XRPD. If a clear solution was obtained, thesolution was left to evaporate at RT. The following solvents, wheretotal amount added is noted in parenthesis immediately after thesolvent, at temperatures of 5° C. and 50° C. were used according to thisprocedure, where the dissolution extent is given within parenthesisafter each temperature which yielded the noted embodiment: Water (100mL) at 5° C. (suspension) and 50° C. (suspension), yielded embodiment 1swhose diffractogram is shown in FIG. 5; methanol (10 mL) at 5° C.(suspension) and at 50° C. (solution), yielded embodiment 1s whosediffractogram is shown in FIG. 5; ethanol (30 mL) at 5° C. (suspension)and at 50° C. (solution), yielded embodiment 1s whose diffractogram isshown in FIG. 5; 2-propanol (30 mL) at 5° C. (suspension) and at 50° C.(solution), yielded embodiment 1s whose diffractogram is shown in FIG.5; 1-propanol (30 mL) at 5° C. (suspension) and at 50° C. (solution),yielded embodiment 1s whose diffractogram is shown in FIG. 5; acetone(100 mL) at 5° C. (suspension) and at 50° C. (solution), yieldedembodiment 1s whose diffractogram is shown in FIG. 5; ethyl acetate (100mL) at 5° C. (suspension) and at 50° C. (turbid), yielded embodiment 1swhose diffractogram is shown in FIG. 5; acetonitrile (100 mL) at 5° C.(suspension) and at 50° C. (solution), yielded embodiment 6 whosediffractogram is shown in FIG. 3; toluene (100 mL) at 5° C. (partiallydissolved) and at 50° C. (turbid), yielded embodiment 1s whosediffractogram is shown in FIG. 5; isopropyl acetate (100 mL) at 5° C.(suspension) and at 50° C. (turbid), yielded embodiment 1s whosediffractogram is shown in FIG. 5; methyl t-butyl ether (100 mL) at 5° C.(suspension) and at 50° C. (suspension), yielded embodiment 1s whosediffractogram is shown in FIG. 5; 2-butanone (100 mL) at 5° C.(suspension) and at 50° C. (solution), yielded embodiment 1s whosediffractogram is shown in FIG. 5; THF (70 mL) at 5° C. (partiallydissolved) and at 50° C. (solution), yielded embodiment 1s whosediffractogram is shown in FIG. 5; DMSO (5 mL) at 5° C. (solution, samplewas frozen and left to evaporate at RT) and at 50° C. (solution),yielded embodiment 1s whose diffractogram is shown in FIG. 5; N-methylpyrrolidinone (5 mL) at 5° C. (solution, left to evaporate at RT) and at50° C. (solution), yielded embodiment 1s whose diffractogram is shown inFIG. 5; diethyl ether (100 mL) at 5° C. (suspension) and at 50° C.(suspension), yielded embodiment 1s whose diffractogram is shown in FIG.5; methyl isobutyl ketone (100 mL) at 5° C. (suspension) and at 50° C.(suspension), yielded embodiment 1s whose diffractogram is shown in FIG.5; DCM (100 mL) at 5° C. (suspension) and at 50° C. (suspension),yielded embodiment 1s whose diffractogram is shown in FIG. 5; heptane(100 mL) at 5° C. (suspension) and at 50° C. (suspension), yieldedembodiment 18 whose diffractogram is shown in FIG. 8; 1-4-dioxane (100mL) at 5° C. (partially dissolved, sample was frozen and left toevaporate at RT) and at 50° C. (suspension), yielded in dried formembodiment 3c whose diffractogram is shown in FIG. 5; nitromethane (100mL) at 5° C. (suspension) and at 50° C. (suspension), yielded a poorlycrystalline embodiment (diffractogram not shown); 1-methoxy-2-propanol(20 mL) at 5° C. (solution) and at 50° C. (solution), yielded in driedform embodiment 20 whose diffractogram is shown in FIG. 11; 2-methyl-THF(100 mL) at 5° C. (suspension) and at 50° C. (suspension), yieldedembodiment 18 whose diffractogram is shown in FIG. 8 and whose TGA andDSC is shown in FIGS. 10A and 10B, respectively; tetralin (100 mL) at 5°C. (suspension) and at 50° C. (turbid), yielded a mixture of embodiment4 and embodiment 1b whose diffractogram is shown in FIG. 3;3-methyl-1-butanol (100 mL) at 5° C. (suspension) and at 50° C.(solution), yielded embodiment 17 whose diffractogram is shown in FIG. 8and whose TGA and DSC is shown in FIGS. 12A and 12B, respectively;anisole (100 mL) at 5° C. (suspension) and at 50° C. (turbid), yielded amixture of embodiment 4 and embodiment 1b whose diffractogram is shownin FIG. 3; t-butanol/water (1:1, 10 mL) at 5° C. (solution) and at 50°C. (solution), yielded in dried form an embodiment 19 whose modulatedDSC is shown in FIG. 9; 1,2-dimethoxyethane (100 mL) at 5° C.(suspension) and at 50° C. (turbid), yielded a mixture of embodiment 4and embodiment 1b whose diffractogram is shown in FIG. 3; cumene (100mL) at 5° C. (suspension) and at 50° C. (turbid), yielded a mixture ofembodiment 4 and embodiment 1b whose diffractogram is shown in FIG. 3;diisopropyl ether (100 mL) at 5° C. (suspension) and at 50° C.(suspension), yielded embodiment 18 whose diffractogram is shown in FIG.8; morpholine (5 mL) at 5° C. (suspension) and at 50° C. (solution),yielded in dried form embodiment 21 whose diffractogram is shown in FIG.11; ethanol:water (95:5, 10 mL) at 5° C. (suspension) and at 50° C.(solution), yielded a poorly crystalline embodiment (diffractogram notshown); ethanol:water (9:1, 5 mL) at 5° C. (solution) and at 50° C.(solution), yielded in dried form embodiment 1s whose diffractogram isshown in FIG. 5; and acetonitrile:water (95:5, 30 mL) at 5° C.(suspension) and at 50° C. (solution), yielded a poorly crystallineembodiment (diffractogram not shown).

Incubation at 5° C.

Several experiments of incubation at 5° C. were performed by treatingembodiment 1s (30 mg) with each solvent, and the mixture was slurried at5° C. for 48 h. An aliquot was taken and immediately analyzed by XRPD.Each aliquot dried for 16 h and was re-analyzed by XRPD. The air-driedsamples were then placed in a vacuum oven (RT) for 24 h before furtheranalysis by XRPD. The following solvents, where total solvent amountadded is noted in parenthesis immediately after the solvent followed bydissolution extent, were used according to this procedure which yieldedthe noted embodiment: Water (30 mL, suspension), yielded embodiment 1swhose diffractogram is shown in FIG. 5; methanol (5 mL, suspension),yielded embodiment 1s whose diffractogram is shown in FIG. 5; ethanol(30 mL, suspension), yielded embodiment 1s whose diffractogram is shownin FIG. 5; 2-propanol (30 mL, suspension), yielded embodiment 1s whosediffractogram is shown in FIG. 5; 1-propanol (30 mL, suspension),yielded embodiment 1s whose diffractogram is shown in FIG. 5; acetone(30 mL, suspension), yielded embodiment 1s whose diffractogram is shownin FIG. 5; ethyl acetate (30 mL, suspension), yielded embodiment 1swhose diffractogram is shown in FIG. 5; acetonitrile (30 mL,suspension), yielded a poorly crystalline embodiment (diffractogram notshown); toluene (30 mL, suspension), yielded embodiment 15 whosediffractogram is shown in FIG. 8; isopropyl acetate (30 mL, suspension),yielded embodiment 17 whose diffractogram is shown in FIG. 8; methylt-butyl ether (30 mL, suspension), yielded embodiment 18 whosediffractogram is shown in FIG. 8; 2-butanone (30 mL, suspension),yielded embodiment 1s whose diffractogram is shown in FIG. 5; THF (30mL, suspension), yielded embodiment 17 whose diffractogram is shown inFIG. 8; diethyl ether (30 mL, suspension), yielded embodiment 1s whosediffractogram is shown in FIG. 5; methyl isobutyl ketone (30 mL,suspension), yielded embodiment 17 whose diffractogram is shown in FIG.8; DCM (30 mL, suspension), yielded embodiment 1s whose diffractogram isshown in FIG. 5; heptane (30 mL, suspension), yielded embodiment 1swhose diffractogram is shown in FIG. 5; 1,4-dioxane (30 mL, suspension),yielded embodiment 3c whose diffractogram from this experiment is shownin FIG. 5; nitromethane (30 mL, suspension), yielded a poorlycrystalline form embodiment 1s whose diffractogram is not shown;propylene glycol (30 mL, suspension), yielded a poorly crystallineembodiment (diffractogram not shown); 2-methyl-tetrahydrofuran (30 mL,suspension), yielded embodiment 18 whose diffractogram is shown in FIG.8; tetralin (30 mL, suspension), yielded a poorly crystalline embodiment1s whose diffractogram is not shown; 3-methyl-1-butanol (30 mL,suspension), yielded embodiment 18 whose diffractogram is shown in FIG.8; anisole (30 mL, suspension), yielded embodiment 1s with an whosediffractogram is similar to that of embodiment 1s (as shown in FIG. 5)except that it displays some additional peaks; 1,2-dimethoxyethane (30mL, suspension), yielded embodiment 1s with an whose diffractogram issimilar to that of embodiment 1s (as shown in FIG. 5) except that itdisplays some additional peaks; cumene (30 mL, suspension), yieldedembodiment 1s with an whose diffractogram is similar to that ofembodiment 1s (as shown in FIG. 5) except that it displays someadditional peaks; diisopropyl ether (30 mL, suspension), yieldedembodiment 17 whose diffractogram is shown in FIG. 8; ethanol:water(95:5, 30 mL, suspension), yielded embodiment 1s whose diffractogram isshown in FIG. 5; acetonitrile:water (95:5, 30 mL, suspension), yielded apoorly crystalline embodiment (diffractogram not shown); andpolyethylene glycol (30 mL, suspension), yielded a poorly crystallineembodiment (diffractogram not shown).

Heat/Cool Maturation

A suspension of embodiment 1s (30 mg) in each solvent was placed in aplatform shaker incubator and subjected to a series of heat-cool cyclesfrom ambient to approximately 50° C. for 24 h. This was achieved byswitching the heating on and off every 4 hours. Shaking was maintainedthroughout. An aliquot from each sample was taken and allowed to air-dryfor 2 h. The air-dried solids were analyzed by XRPD, then vacuum driedusing a vacuum oven (RT, 24 h) and were re-analyzed by XRPD. Each sampleobtained in this experiment was vacuum dried and after vacuum dryingeach sample was analyzed by XRPD incubation at elevated temperature. Thefollowing solvents, where total solvent amount added is noted inparenthesis immediately after the solvent, were used according to thisprocedure which yielded the noted embodiment: Water (20 mL) yieldedembodiment 1s whose diffractogram is shown in FIG. 5; methanol (5 mL)yielded embodiment 22 whose diffractogram is shown in FIG. 13; ethanol(5 mL) yielded embodiment 1s whose diffractogram is shown in FIG. 5;2-propanol (10 mL) yielded embodiment 27 whose diffractogram for thisexperiment is shown in FIG. 13; 1-propanol (10 mL) yielded embodiment 23whose diffractogram is shown in FIG. 13; acetone (20 mL) yieldedembodiment 1s whose diffractogram is shown in FIG. 5; ethyl acetate (20mL) yielded a poorly crystalline form of embodiment 1s whosediffractogram is not shown; acetonitrile (20 mL) yielded a poorlycrystalline embodiment 24 whose diffractogram is shown in FIG. 13;toluene (20 mL) yielded a poorly crystalline embodiment 1s whosediffractogram is not shown; isopropyl acetate (20 mL) yielded embodiment18 whose diffractogram is shown in FIG. 13; methyl t-butyl ether (20 mL)yielded a poorly crystalline embodiment 1s whose diffractogram is notshown; 2-butanone (20 mL) yielded embodiment 26 whose diffractogram isshown in FIG. 13; THF (20 mL) yielded embodiment 18 whose diffractogramis shown in FIG. 13; diethyl ether (20 mL) yielded a poorly crystallineembodiment 1s whose diffractogram is not shown; methyl isobutyl ketone(20 mL) yielded embodiment 25 whose diffractogram is shown in FIG. 13;DCM (20 mL) yielded a poorly crystalline form of embodiment 1s whosediffractogram is not shown; heptane (20 mL) yielded embodiment 1s whosediffractogram is shown in FIG. 5; 1,4-dioxane (20 mL) yielded embodiment27 whose diffractogram for this experiment is shown in FIG. 13;nitromethane (20 mL) yielded a poorly crystalline embodiment 1s whosediffractogram is not shown; propylene glycol (5 mL) yielded a poorlycrystalline embodiment (diffractogram not shown);2-methyl-tetrahydrofuran (20 mL) yielded embodiment 18 whosediffractogram is shown in FIG. 13; tetralin (20 mL) yielded embodiment1s whose diffractogram is shown in FIG. 5; 3-methyl-butanol (20 mL)yielded embodiment 18 whose diffractogram is shown in FIG. 13; anisole(20 mL) yielded embodiment 16 whose diffractogram is shown in FIG. 13and whose TGA and DSC are shown in FIGS. 23A and 23B, respectively;1,2-dimethoxyethane (20 mL) yielded embodiment 29 whose diffractogram isshown in FIG. 13; cumene (20 mL) yielded embodiment 1s whosediffractogram is shown in FIG. 5; diisopropyl ether (20 mL) yieldedembodiment 17 whose diffractogram is shown in FIG. 13; ethanol:water(95:5, 20 mL) yielded embodiment 30 whose diffractogram is shown in FIG.13; acetonitrile:water (95:5, 20 mL) yielded a poorly crystalline formof embodiment 1s whose diffractogram is not shown; and polyethyleneglycol (5 mL) yielded embodiment 31 whose diffractogram is shown in FIG.13.

Incubation of Embodiment 1s at 60° C.

Embodiment 1s (30 mg) was treated with solvent and shaken at 60° C. for24 h. An aliquot was taken out and allowed to air-dry for 16 h. Thedried samples were then analyzed by XRPD. The following solvents, wheretotal solvent amount added is noted in parenthesis immediately after thesolvent, were used according to this procedure which yielded the notedembodiment: Water (10 mL) yielded embodiment 1s whose diffractogram isshown in FIG. 5; ethanol (10 mL) yielded embodiment 32 whosediffractogram is shown in FIG. 14; 2-propanol (10 mL) yielded embodiment33 whose diffractogram is shown in FIG. 14; 1-propanol (10 mL) yieldedembodiment 23 whose diffractogram is shown in FIG. 14; acetone (10 mL)yielded embodiment 1s whose diffractogram is shown in FIG. 5; ethylacetate (10 mL) yielded embodiment 34 whose diffractogram is shown inFIG. 14; acetonitrile (10 mL) yielded embodiment 35 whose diffractogramis shown in FIG. 14; toluene (10 mL) yielded embodiment 36 whosediffractogram is shown in FIG. 14; isopropyl acetate (10 mL) yieldedembodiment 25 whose diffractogram for this experiment is shown in FIG.14; methyl t-butyl ether (10 mL) yielded embodiment 35 whosediffractogram is shown in FIG. 14; 2-butanone (10 mL) yielded embodiment38 whose diffractogram is shown in FIG. 14; THF (10 mL) yieldedembodiment 33 whose diffractogram for this experiment is shown in FIG.14; diethyl ether (10 mL) yielded embodiment 1s whose diffractogram isshown in FIG. 5; methyl isobutyl ketone (10 mL) yielded embodiment 25whose diffractogram for this experiment is shown in FIG. 14; DCM (10 mL)yielded embodiment 1s whose diffractogram is shown in FIG. 5; heptane(10 mL) yielded embodiment 1s whose diffractogram is shown in FIG. 5;1-4-dioxane (10 mL) yielded embodiment 33 whose diffractogram is shownin FIG. 14; nitromethane (10 mL) yielded embodiment 1s whosediffractogram is shown in FIG. 5; propylene glycol (10 mL) yieldedembodiment 28 whose diffractogram for this experiment is shown in FIG.14; 2-methyl-tetrahydrofuran (10 mL) yielded embodiment 33 whosediffractogram is shown in FIG. 14; tetralin (10 mL) yielded a mixture(diffractogram of the mixture not shown) of embodiment 1s whosediffractogram is shown in FIG. 5 and embodiment 19 whose modulated DSCprofile is shown in FIG. 9; 3-methyl-1-butanol (10 mL) yieldedembodiment 33 whose diffractogram is shown in FIG. 14; anisole (10 mL)yielded embodiment 36 whose diffractogram is shown in FIG. 14;1,2-dimethoxyethane (10 mL) yielded embodiment 34 whose diffractogram isshown in FIG. 14; cumene (10 mL) yielded embodiment 1s whosediffractogram is shown in FIG. 5; diisopropyl ether (10 mL) yieldedembodiment 17 whose diffractogram is shown in FIG. 8; ethanol:water(95:5, 10 mL) yielded embodiment 28 whose diffractogram is shown in FIG.14; and polyethylene glycol (5 mL) yielded embodiment 39 whosediffractogram for this experiment is shown in FIG. 14

High Temperature Maturation

Each of a plurality of embodiment 19 (25 mg) samples was treated with anamount of a solvent as indicated below yielding in turn a plurality ofsamples, each agitated at 60° C. for 24 h.

Solids from each sample were isolated, air-dried for 16 h and analyzedby XRPD. The following solvents, where total solvent amount added isnoted in parenthesis immediately after the solvent followed bydissolution extent, were used according to this procedure which yieldedthe noted embodiment: Water (125 μL, suspension) yielded embodiment 1swhose diffractogram is shown in FIG. 5; methanol (125 μL, suspension)yielded embodiment 1s whose diffractogram is similar to thediffractogram for embodiment 1s shown in FIG. 5 except that it displayssome additional peaks; ethanol (125 μL, suspension) yielded embodiment1s whose diffractogram is shown in FIG. 5; 2-propanol (75 μL,suspension) yielded embodiment 37 whose diffractogram is shown in FIG.15; 1-propanol (75 μL, suspension) yielded embodiment 40 whosediffractogram is shown in FIG. 15; acetone (75 μL, suspension) yieldedembodiment 1s whose diffractogram is shown in FIG. 5; ethyl acetate (75μL, suspension) yielded embodiment 1s whose diffractogram is shown inFIG. 5; acetonitrile (75 μL, suspension) yielded embodiment 1s whosediffractogram is shown in FIG. 5; toluene (75 μL, suspension) yieldedembodiment 1s whose diffractogram is shown in FIG. 5; isopropyl acetate(75 μL, suspension) yielded embodiment 37 whose diffractogram is shownin FIG. 15; methyl t-butyl ether (75 μL, suspension), yielded embodiment33 whose diffractogram is shown in FIG. 14; 2-butanone (75 μL,suspension) yielded embodiment 1s whose diffractogram is shown in FIG.5; THF (75 μL, suspension) yielded embodiment 37 whose diffractogram isshown in FIG. 15; diethyl ether (150 μL, suspension) yielded embodiment1s whose diffractogram is shown in FIG. 5; methyl isobutyl ketone (150μL, suspension) yielded embodiment 33 whose diffractogram is shown inFIG. 14; DCM (75 μL, suspension) yielded embodiment 1s whosediffractogram is shown in FIG. 5; heptane (150 μL, suspension) yieldedembodiment 41 whose diffractogram is shown in FIG. 15; 1,4-dioxane (75μL, suspension) yielded embodiment 3c whose diffractogram is shown inFIG. 5; nitromethane (75 μL, suspension) yielded embodiment 42 whosediffractogram is shown in FIG. 15; propylene glycol (75 μL, suspension)yielded embodiment 43 whose diffractogram is shown in FIG. 15;2-methyl-tetrahydrofuran (150 μL, suspension) yielded embodiment 33whose diffractogram is shown in FIG. 14; tetralin (150 μL, suspension),yielded embodiment 33 whose diffractogram is shown in FIG. 14;3-methyl-1-butanol (75 μL, suspension) yielded embodiment 33 whosediffractogram is shown in FIG. 14; anisole (150 μL, suspension) yieldedembodiment 1s whose diffractogram is shown in FIG. 5;1,2-dimethoxyethane (75 μL, suspension) yielded embodiment 1s whosediffractogram is shown in FIG. 5; cumene (150 μL, suspension) yieldedembodiment 44 whose diffractogram is shown in FIG. 15; diisopropyl ether(150 μL, suspension) yielded embodiment 33 whose diffractogram is shownin FIG. 14; ethanol:water (95:5, 75 μL, suspension) yielded embodiment45 whose diffractogram is shown in FIG. 15; acetonitrile:water (95:5, 75μL, suspension) yielded embodiment 1s whose diffractogram showed cellexpansion when compared to the diffractogram shown in FIG. 5; andpolyethylene glycol (75 μL, suspension) yielded embodiment 46 whosediffractogram is shown in FIG. 15.

Thermocycling

Each of a plurality of embodiment 19 (25 mg) samples was treated with anamount of a solvent as indicated below yielding in turn a plurality ofsamples, each sample was matured by thermocycling (40° C.-60° C., 4 hcycles) for 24 h. Solids were isolated, air-dried for 16 h and analyzedby XRPD. The following solvents, where total solvent amount added isnoted in parenthesis immediately after the solvent followed by theobserved appearance at 24 hours, were used according to this procedurewhich yielded the noted embodiment: Water (125 μL, green tinge solid)yielded embodiment 1s whose diffractogram is shown in FIG. 5; methanol(75 μL, transparent solid) yielded embodiment 11 whose diffractogramshowed peaks that were shifted at high angle when compared to thediffractogram in FIG. 7; ethanol (100 μL, green tinge solid) yieldedembodiment 1s whose diffractogram is shown in FIG. 5; 2-propanol (75 μL,yellow tinge solid) yielded embodiment 33 whose diffractogram is shownin FIG. 14; 1-propanol (75 μL, white suspension) yielded embodiment 33whose diffractogram is shown in FIG. 14; acetone (75 μL, green tingesolid) yielded embodiment 47 whose diffractogram is shown in FIG. 16;ethyl acetate (75 μL, white suspension) yielded embodiment 33 whosediffractogram is shown in FIG. 14; acetonitrile (75 μL, whitesuspension) yielded a poorly crystalline of embodiment 6 whosediffractogram is shown in FIG. 3; toluene (75 μL, transparent solid)yielded embodiment 36 whose diffractogram is shown in FIG. 14; isopropylacetate (75 μL, white solid) yielded embodiment 33 whose diffractogramis shown in FIG. 14; methyl t-butyl ether (75 μL, white suspension)yielded embodiment 6 whose diffractogram is shown in FIG. 3; 2-butanone(75 μL, off-white solid) yielded embodiment 33 whose diffractogram isshown in FIG. 14; THF (75 μL, off-white solid) yielded embodiment 48whose diffractogram is shown in FIG. 16; diethyl ether (150 μL,off-white solid) yielded embodiment 49 whose diffractogram is shown inFIG. 16; methyl isobutyl ketone (150 μL, off-white solid) yieldedembodiment 25 whose diffractogram is very similar to the diffractogramfor embodiment 25 that is shown in FIG. 13; DCM (125 μL, whitesuspension) yielded embodiment 1s whose diffractogram is shown in FIG.5; heptane (150 μL, white solid) yielded embodiment 19 whose modifiedDSC profile is shown in FIG. 9; 1,4-dioxane (75 μL, white solid) yieldedembodiment 3c whose diffractogram is shown in FIG. 5; nitromethane (75μL, white suspension) yielded embodiment 50 whose diffractogram is shownin FIG. 16; propylene glycol (75 μL, cream suspension) yieldedembodiment 10 whose diffractogram is very similar to the diffractogramfor embodiment 10 (as shown in FIG. 16), except that it shows anamorphous halo; 2-methyl-tetrahydrofuran (150 μL, white solid) yieldedembodiment 48 whose diffractogram is shown in FIG. 16; tetralin (150 μL,white solid) yielded a poorly crystalline embodiment whose diffractogramis not shown; 3-methyl-1-butanol (75 μL, white suspension) yieldedembodiment 25 whose diffractogram is shown in FIG. 13; anisole (150 μL,white suspension) yielded embodiment 51 whose diffractogram is shown inFIG. 16; 1,2-dimethoxyethane (75 μL, white suspension) yieldedembodiment 52 whose diffractogram is shown in FIG. 16; cumene (150 μL,white solid) yielded a poorly crystalline embodiment whose diffractogramis not shown; diisopropyl ether (150 μL, white solid) yielded embodiment6 whose diffractogram is shown in FIG. 3; ethanol:water (95:5, 75 μL,transparent solid) yielded embodiment 11 whose diffractogram is shown inFIG. 7; acetonitrile:water (95:5, 75 μL, transparent solid) yieldedembodiment 53 whose diffractogram is shown in FIG. 16; and propyleneglycol (75 μL, pale pink suspension) yielded embodiment 31 whosediffractogram is very similar to the diffractogram for embodiment 31 (asshown in FIG. 13), except that it shows an amorphous halo.

Any one of embodiments 11, 11b, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 and 53 of compound Ex. 1 andany combination thereof is an embodiment of compounds according to thisinvention. Still other embodiments of compounds according to thisinvention include compound Ex. 1 as a non-hygroscopic solvate, such asembodiment 11 of compound Ex. 1. Still other embodiments of compoundsaccording to this invention include compound Ex. 1 in amorphous form,such as embodiment 19 of compound Ex. 1. Any one of embodiments 11, 16,17, and 18 of compound Ex. 1 and any combination thereof is anembodiment of compounds according to this invention. Further embodimentsof this invention include compounds according to this invention in theform of pharmaceutically acceptable co-crystals. Additional embodimentsof this invention include compounds according to this invention in theform of pharmaceutically acceptable salts.

Examples 1-12 are JAK inhibitors and were tested in enzymatic andcellular assays. The results of the enzymatic assay are presented inTable 4 which is entitled Results of Enzymatic Inhibition Assays.Examples 1-12 were also tested in three cellular assays: IL-2 pSTAT5(JAK1/JAK3), IFNα pSTAT4 (JAK1/TYK2) and GM-CSF pSTAT5 (JAK2/JAK2) withthe results presented in Table 5 entitled Cell-Based Assay Data. Belowis the description of how the enzymatic assay was performed includingthe materials used in the assay (under the heading Materials), how theassay was set up (under the heading Assay protocol), and the method usedto analyze the data (under the heading High-throughput Mass Spectrometry(HTMS) Method).

Enzymatic Inhibition Assay

Materials

Substrate (NH2-KGGEEEEYFELVKK-CO2), internal standard peptide(NH2-SWGAIETDKEYYTVKD-CO2) and product peptide (for standard curve only)(NH2-KGGEEEEY-Pi-FELVKK-CO2), were purchased from AnaSpec (Fremont,Calif., USA). JAK1-JH1JH2 (574-1154 with a His-GST Tag and a C-terminaltev (ENLYFQ-G) cleavage site), JAK3-JH1JH2 (512-1124 with a GST Tag anda C-terminal tev (ENLYFQ-G) cleavage site), and Tyk2-JH1JH2(8H_tev_580-1182-C936A-C1142A with a C-terminal tev (ENLYFQ-G) cleavagesite) were purified internally. JAK2-JH1JH2 (532-1132 with a GST tag andC-terminal tev (ENLYFQ-G) cleavage site), was purchased from Invitrogen.LC/MS grade water and acetonitrile (ACN), were purchased from HoneyWell,Burdick & Jackson (Muskegon, Mich., USA). Dimethylsulfoxide 99.8% (DMSO)and trifluoroacetic acid 99.5% (TFA) were purchased from EMD Chemical(Gibbstown, N.J., USA). Adenosine triphosphate (ATP),4-morpholinepropanesulfonic acid (MOPS), magnesium chloride (MgCl₂),ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), formicacid >95% (FA) and Tween-20 were purchased from Sigma (St Louis, Mo.,USA). 384-well polypropylene plates, Cat #781280 were purchased fromGreiner (Monroe, N.C.), RapidFire™ cartridge A C4 Column (AgilentTechnologies, Santa Clara, Calif.).

The HTMS experiments were performed in positive ionization mode on aRapidFire 300 instrument (Agilent Technologies, Santa Clara, Calif.),coupled with an ABSiex QTrap 4000 system with an Electrospray Ionizationsource (RF-MS) (Concord, ON, Canada). The RapidFire system was run with3 Agilent 1200 series isocratic pumps Agilent Technologies (Santa Clara,Calif.) and one peristaltic pump model ISM832C from Ismatec (Wertheim,Germany). The entire system was operated using the RapidFire softwareinterfaced with Analyst software for the mass spectrometer.

Assay Protocol

11-point dosing series were made for each compound by serially diluting1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serialdilution plates, sample was transferred to a 384 wells assay plate(#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.),or Biosero ATS (San Diego, Calif.). The compounds were tested induplicate. Column 12 was used for positive controls, and column 24contained negative controls with no enzyme added. A compound from ourinternal collection, with inhibitory activity for JAK isoforms, was usedas a reference compound. The final concentration of DMSO was ≤0.25% in a20 μL reaction. Assay conditions for each of the proteins are summarizedin Table 3. The enzyme reaction was initiated by the addition of 10 μLof enzyme and ATP mixture to 10 μL of substrate solution prepared inreaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl₂, 1 mM EDTA, 2 mM DTT,0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30min prior to the reaction initiation. Immediately after the addition ofthe enzyme to the reaction mixture, the plate was centrifuged at 1000rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK3 and 90minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by theaddition of 20 μL of 0.5% TFA containing 0.15 μM of internal standardpeptide using Multidrop Combi reagent dispenser (Thermo Scientific,Waltham, Mass.). Several wells in column 24 were typically used for theproduct standard curve. After the quench, the assay plate wascentrifuged at 3000 rpm for 3 minutes and sealed with pierceablealuminum foil (Cat #06644-001, Agilent) using a PlateLoc (AgilentTechnologies, Santa Clara, Calif.). The plates then were transferred onto the RapidFire for the MS analysis. Compound inhibition was assessedby a decrease of the phosphorylated product levels in sample wellscompared to the non-inhibited enzyme reaction. The assay conditions forthe above assays are shown in Table 3 and the results of Ex. 1-12 astested in these assays are shown in Table 4.

TABLE 3 Assay conditions for JAK family enzyme assays* [enzyme], [ATP],[Substrate], [IS], Enzyme nM μM μM nM JAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 30 40 100 3.6 JAK3-JH1JH2 2.0 150 40 100 Tyk2-JH1JH225 or 50 200 100 14.7 *Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl₂,1 mM EDTA, 2 mM DTT, 0.002% Tween-20; “IS” stands for internal standardpeptide; “Substrate” stands for peptide.

High-Throughput Mass Spectrometry (HTMS) Method

The sample analysis on the RapidFire was performed using a mobile phaseA1 consisting of Water/TFA/FA (100:0.01:0.1, v/v/v), a mobile phase B 1consisting of ACN/Water/TFA/FA (80:20:0.01:0.1, v/v/v). The followingrun parameters were used: state 1 (aspirate), 250 ms; state 2(load/wash), 3000 ms; state 3 (elute), 4000 ms; state 4 (re-equlibrate),1000 ms with a flow rate of 1.25 mL/min. The samples were aspirateddirectly from the 384-well assay plate and delivered onto RF-MSmicroscale solid-phase C4 extraction cartridge (Type A). The undesiredcomponent such as salt, cofactor, detergent and large protein werewashed out and the retained analytes (substrate, product and IS) werecoeluted directly onto the ABSiex Qtrap 4000 system. The quantificationof peptide (substrate), phospho-peptide (product) and internal standardpeptide (IS) was performed by MRM using 562→136.0, 589.2→215.7 and953.2→158.8 (or 974.2→158.8) transitions respectively.

TABLE 4 Results of Enzymatic Inhibition Assays Test JAK1_JH1JH2JAK2_I_JH1JH2 JAK3_I_JH1JH2 Tyk2_I_JH1JH2 Compound IC₅₀ (nM) IC₅₀ (nM)IC₅₀ (nM) IC₅₀ (nM) A <0.2 <0.2 12.4 0.9 B <0.2 <0.2 13.4 <0.2 C <0.20.6 49.7 0.2 Ex. 1 0.4 8.6 92.2 7.4 Ex. 2 0.2 1.0 33.9 1.5 Ex. 3 0.2 6.282.8 11.6 Ex. 4 0.1 6.6 96.2 2.2 Ex. 5 0.3 2.1 23.1 4.1 Ex. 6 0.1 1.428.2 1.1 Ex. 7 0.2 5.6 98.4 4.8 Ex. 8 0.4 7.0 75.6 6.6 Ex. 9 0.2 6.679.9 4.7 Ex. 10 1.0 6.5 87.5 9.0 Ex. 11 0.4 1.6 30.3 1.5 Ex. 12 0.9 9.4101.3 8.7

Cellular Assays

IL-2 pSTAT5 (JAK1/JAK3) Cellular Assay

The AlphaLISA assay (based on Alpha Technology from PerkinElmer) wasperformed by first plating freshly thawed PBMCs (Biological SpecialtyCorporation) in 384-well plates at 30,000 cells per 4 μL per well inHBSS (Hanks' Balanced Salt Solution) containing 0.1% IgG (immunoglobulinG)-free, protease-free BSA (bovine serum albumin) (JacksonImmunoResearch Cat. No. 001-000-161). The cells were then treated with 2μL/well of compounds diluted in DMSO at half-log titratedconcentrations, with a highest test concentration of 10 M and 0.5% finalDMSO concentration, for thirty minutes at 37° C. Next, the cells werestimulated with 2 μL/well of IL-2 (R&D Systems Cat. No. 202-IL-050) at 5ng/mL for thirty minutes at 37° C. The cellular reactions wereterminated by the addition of 2 μL/well of lysis buffer (PerkinElmerCat. No. ALSU-PST5S-A10K) followed by an incubation of five minutes atroom temperature. 5 μL/well of acceptor mix (PerkinElmer Cat. No.ALSU-PST5-A10K) was added to the cells and incubated in the dark for onehour at room temperature. Then, 5 μL/well of donor mix (PerkinElmer Cat.No. ALSU-PST5-A10K) was added to the cells and incubated in the darkovernight at room temperature. Finally, the plates were read on aPerkinElmer EnVision for detection of the time-resolved fluorescencesignal. The percentage of IL-2-dependent pSTAT5 inhibition wasdetermined at the compound test concentrations; and for each compound, adose curve was generated and the IC₅₀ was calculated. Compound IC₅₀ wascalculated by nonlinear regression, sigmoidal dose response analysis ofthe half-log dilution titration curve of the compound concentration vs.Alpha signal. The acronym “Alpha” stands for amplified luminescentproximity homogeneous assay; the Alpha signal is aluminescent/fluorescent signal.

IFNα pSTAT4 (JAK1/TYK2) Cellular Assay

The AlphaLISA assay (based on Alpha Technology from PerkinElmer) wasperformed by first plating freshly thawed PBMCs (Biological SpecialtyCorporation) in 384-well plates at 100,000 cells per 6 μL per well inDMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovineserum) and 1,000 I.U./mL penicillin and 1,000 μg/mL streptomycin. Thecells were then treated with 2 μL/well of compounds diluted in DMSO athalf-log titrated concentrations, with a highest test concentration of10 μM and 0.5% final DMSO concentration, for thirty minutes at 37° C.Next, the cells were stimulated with 2 μL/well of IFNα (PBL AssayScience Cat. No. 11101-2) at 4 ng/mL for thirty minutes at 37° C. Thecellular reactions were terminated by the addition of 2 μL/well of lysisbuffer (PerkinElmer Cat. No. ALSU-PST4-A1 (K) followed by an incubationof five minutes at room temperature. 4 μL/well of acceptor mix(PerkinElmer Cat. No. ALSU-PST4-A10K) was added to the cells andincubated in the dark for one hour at room temperature. Then, 4 μL/wellof donor mix (PerkinElmer Cat. No. ALSU-PST4-A10K) was added to thecells and incubated in the dark overnight at room temperature. Finally,the plates were read on a PerkinElmer EnVision for detection of thetime-resolved fluorescence signal. The percentage of IFNα-dependentpSTAT4 inhibition was determined at the compound test concentrations;and for each compound, a dose curve was generated and the IC₅₀ wascalculated. Compound IC₅₀ was calculated by nonlinear regression,sigmoidal dose response analysis of the half-log dilution titrationcurve of the compound concentration vs. Alpha signal. The term “Alpha”is defined in the immediately preceding cellular assay description.

GM-CSF pSTAT5 (JAK2/JAK2) Cellular Assay

The AlphaLISA assay (based on Alpha Technology from PerkinElmer) wasperformed by first plating freshly thawed PBMCs (Biological SpecialtyCorporation) in 384-well plates at 30,000 cells per 4 μL per well inHBSS containing 0.1% IgG-free, protease-free BSA (Jackson ImmunoResearchCat. No. 001-000-161). The cells were then treated with 2 μL/well ofcompounds diluted in DMSO at half-log titrated concentrations, with ahighest test concentration of 10 μM and 0.5% final DMSO concentration,for thirty minutes at 37° C. Next, the cells were stimulated with 2μL/well of GM-CSF (R&D Systems Cat. No. 215-GM-050) at 11 pg/mL forfifteen minutes at 37° C. The cellular reactions were terminated by theaddition of 2 μL/well of lysis buffer (PerkinElmer Cat. No.ALSU-PST5-A10K) followed by an incubation of five minutes at roomtemperature. 5 μL/well of acceptor mix (PerkinElmer Cat. No.ALSU-PST5-A10K) was added to the cells and incubated in the dark for onehour at room temperature. Then, 5 μL/well of donor mix (PerkinElmer Cat.No. ALSU-PST5-A10K) was added to the cells and incubated in the darkovernight at room temperature. Finally, the plates were read on aPerkinElmer EnVision for detection of the time-resolved fluorescencesignal. The percentage of GM-CSF-dependent pSTAT5 inhibition wasdetermined at the compound test concentrations; and for each compound, adose curve was generated and the IC₅₀ was calculated. Compound IC₅₀ wascalculated by nonlinear regression, sigmoidal dose response analysis ofthe half-log dilution titration curve of the compound concentration vs.Alpha signal. The term “Alpha” is defined in the IL-2 pSTAT5 (JAK1/JAK3)cellular assay description.

TABLE 5 Cell-Based Assay Data IL-2 IFNα GM-CSF pSTAT5 pSTAT4 pSTAT5 Test(JAK1/JAK3) (JAK1/TYK2) (JAK2/JAK2) Compound IC₅₀ (nM) IC₅₀ (nM) IC₅₀(nM) Ex. 1 21.6 59.5 83.9 Ex. 2 9.0 20.8 61.0 Ex. 3 6.4 10.1 21.9 Ex. 435.5 64.7 119.4 Ex. 5 6.4 38.1 28.9 Ex. 6 6.7 39.4 25.1 Ex. 7 9.7 38.567.3 Ex. 8 20.6 42.8 33.8 Ex. 9 11.2 35.9 26.9 Ex. 10 16.8 40.4 44.6 Ex.11 49.1 96.5 201.5 Ex. 12 13.9 81.4 75.4

Examples 1-12 were tested in solubility and permeability assays. Theresults of the solubility assay are presented in Table 6 which isentitled Solubility Assay Data and the results of the permeability assayare presented in Table 7 entitled MDCK-MDR1 Permeability Data. Thesesolubility and permeability assays are described below under theheadings Solubility Assays and Permeability Assays, respectively.

Solubility Assays

Solubility measurements were conducted in the following solubilitymedia: Simulated gastric (34.2 mM of sodium chloride and 100 mM ofhydrochloric acid) or simulated intestinal fluids (fasted state [pH6.5]: 3 mM of sodium taurocholate, 0.75 mM of lecithin, 28.4 mM ofmonobasic sodium phosphate, 8.7 mM of sodium hydroxide, and 105.9 mM ofsodium chloride). Test compounds were dissolved in DMSO at aconcentration of 10 mM. The test compounds were dispensed (20 μL) intoNunc 1-mL-96-Deep-Well-PP plates, and the DMSO was evaporated vianitrogen blow down from a TurboVap 96 for 6 hours or until a dry residuewas produced. Then, 400 μL of solubility media was added to the wellcontaining the dry solid. A Pre-Slit Well Cap was securely placed overthe well plate block, and the samples were vigorously stirred for 2-5days at ambient temperature. After the incubation period, the sampleswere filtered through an AcroPrep 1-mL-96-Filter plate into a new2-mL-96-Deep-Well-PP plate, and the supernatants were quantified byUV-HPLC using a 3-point calibration ranging from 0.004-0.55 mM. Thesolubility for each compound was calculated from the following equation:

${Solubility} = {\frac{{Sample}\mspace{14mu} {Peak}\mspace{14mu} {Area}}{{Average}\mspace{20mu} {Response}\mspace{14mu} {Factor}\mspace{14mu} {from}\mspace{14mu} 3\mspace{14mu} {Standards}}.}$

The solubility values were in the range of 4-400 μM. Values outside ofthis range were reported as either <4 μM or >400 M. Solubilities arereported as long as the compound under study was sufficiently stable tocomplete the corresponding solubility determination.

TABLE 6 Solubility Assay Data Test SGF solubility SIF solubilityCompound (μM) (μM) A >400 >400 B >400 75 C >400 >400 Ex. 1 >400 387 Ex.2 >400 >400 Ex. 3 >400 >400 Ex. 4 >400 >400 Ex. 5 >400 198 Ex.6 >400 >400 Ex. 7 >400 81 Ex. 8 >400 >400 Ex. 9 >400 >400 Ex. 10 >400359 Ex. 11 >400 >400 Ex. 12 >400 >400

Permeability Assays

Permeability measurements were conducted according to the Cyprotexprotocol using the MDCK-MDR1 cell line obtained from the NIH (Rockville,Md., USA). Cells between passage numbers 6-30 were seeded onto aMultiscreen Plate™ (Millipore) at a cell density of 3.4×10⁵ cells/cm²and cultured for three days before permeability studies were conducted.The cells in this assay form a cohesive sheet of a single cell layerfiling the surface area of the culture dish, also known as a confluentmonolayer, and on day four the test compound was added to the apicalside of the membrane and the transport of the compound across themonolayer was monitored over a time period of 60 min.

In a simple and basic way of introducing “A” and “B” terms that areoften used in these assays, the apical (“A”) side or compartment of anentity is the side of such entity that is exposed to the lumen orexterior environment, whereas the basolateral (“B”) side or compartmentis the side or compartment of such entity that is exposed to thetypically internal environment, encompassing the opposite side. Forexample, when such entity is illustratively an intestinal epitheliumcell, the apical side of such intestinal cell would be the side of thecell exposed to the intestinal lumen, whereas the basolateral side wouldbe the side that is exposed to the blood.

Test compounds were dissolved in DMSO at a concentration of 10 mM. Thedosing solutions were prepared by diluting test compound with assaybuffer (Hanks Balanced Salt Solution), pH 7.4, at a final concentrationof 5 μM. For assessment of apical to basolateral (“A-B”) permeability,buffer was removed from the apical compartment and replaced with testcompound dosing solution with or without the permeability glycoprotein(“PgP”, “P-gP”, “Pgp” or “P-gp”) inhibitor elacridar (2 μM). Forassessment of basolateral to apical (“B-A”) permeability, buffer wasremoved from the companion plate and replaced with test compound dosingsolution. Incubations were carried out in duplicate at 37° C. in anatmosphere of 5% CO2 with a relative humidity of 95%. Each assayincluded the reference markers propranolol (high permeability) andprazosin (PgP substrate). After incubation for 60 minutes, apical andbasolateral samples were diluted and test compounds quantified byLC/MS/MS using an 8-point calibration in the range 0.0039 to 3 μM withappropriate dilution of the samples (receiver dilution factor=1; donorand Co dilution factor=10). The permeability coefficient (P_(app)) foreach compound was calculated from the following equation:P_(app)=(dQ/dt)/(C₀×S), where dQ/dt is the rate of permeation of thedrug across the cells, C₀ is the donor compartment concentration at timezero, and S is the area of the cell monolayer.

The percent recovery was measured for all incubation conditions. Thesemeasurements did not reveal unacceptable compound/plate binding orcompound accumulation in the cell monolayer.

The second and third columns in Table 7 show the values of P_(app(A−B))for the apical-to-basolateral compound transport without (second column)and with a P-gp inhibitor (third column, noted as P^(e) _(app(A−B)))that was elacridar. P_(app(A−B)) gives an indication of permeationextent across the cells in this assay, which is envisaged to model thetranscellular transport across Pgp-expressing cells, such asPgp-expressing gastrointestinal tract cells. P^(e) _(app(A−B)) values(P_(app(A−B)) in the presence of the P-gp inhibitor) given in column 3are determined to confirm the role of P-gp in the compound efflux. Thefourth column in Table 7 shows the values of P_(app(B−A)) for thebasolateral-to-apical compound transport. Test compound efflux ratiosare given in the fifth column of Table 7 as P_(app(B−A))/P_(app(A−B)) byusing the corresponding permeability coefficient values from the fourthand second columns in the same table. The efflux ratios (fifth column,Table 7) are consistently greater than 2 for compounds (A)-(C) and alsofor compounds Ex. 1-12, which indicates that compound efflux occurs forall such compounds.

P_(app(A−B)) values in column 2 are generally low and comparable forreference compounds (A)-(C) and also for compounds Ex. 1-12. These lowvalues indicate low permeability for all such compounds, which is due tothe P-gp effects since all such compounds are P-gp substrates asindicated by the values given in column 5 being all greater than 2. Tobe characterized as having low permeability, the values given in thethird and fourth columns for P^(e) _(app(A−B)) and P_(app(B−A)),respectively, should be low. However, these data show that theP_(app(B−A)) values for compounds (A)-(C) are greater than thecorresponding values compounds Ex. 1-12.

The integrity of each monolayer was monitored by examining thepermeation of lucifer yellow by fluorimetric analysis. This examinationrevealed that the cells in this assay maintained a satisfactoryconfluent monolayer.

TABLE 7 MDCK-MDR1 Permeability Data MDCK- MDCK- MDCK- MDR1 MDR1 MDR1Test P_(app(A-B)) P^(e) _(app(A-B)) P_(app(B-A)) Com- (10⁻⁶ cm/ (10⁻⁶cm/ (10⁻⁶ cm/ P_(app(B-A))/ pound** sec) @ 5 (μM) sec) @ 5 (μM) sec) @ 5(μM) P_(app(A-B)) A 1.3 22   55.3  43 B 0.4 1.7 23.5  59 C 0.5 2.5 23.1 46 Ex. 1 <0.5, 0.4  <0.5, 1.1  0.9, 1.1 >1.9, 3.3  Ex. 2* 1.1 1.6 4.84.4 Ex. 3 <0.4, <0.5 2.3, 1.6 17, 16 >41, >33 Ex. 4 0.1 0.5 1.3 8.7 Ex.5 <0.4, <0.5 0.7, 0.5 1.8, 2.1 >4.8, >4.5 Ex. 6 <0.3, <0.3 0.9, 1.1 2.5,2.6 >8.4, >8.7 Ex. 7 <0.4  1.2 3.6 >9 Ex. 8 0.1 0.5 1.7 11.5 Ex. 9 <0.4 0.6 1.8 >4.2 Ex. 10 <0.4  1.1 7.1 >16.9 Ex. 11 <0.4  0.8 1.1 >2.9 Ex. 12<0.5  0.6 1.1 >2.2 *Starting concentration was measured to be >7 μM forA to B, A to B (with elacridar), and B to A conditions. **Unlessindicated otherwise, compounds (A)-(C) and Ex. 1-12 were tested at aconcentration of 5 μM. For data shown in cells with two data points,compounds were tested twice.

In Vivo Studies

Oral Dosing—Protocol 1

Three non-fasted female C57BL/6 mice were orally administered testcompound at a dose of 25 mg/kg p.o. as a solution in 20%hydroxypropyl-beta-cyclodextrin (HPβCD) at a dose volume of 5 mL/kg.Blood samples were collected at 0.5, 2, and 4 h post dose viaretro-orbital bleed or venipuncture of the dorsal metatarsal vein. Bloodsamples were collected into tubes containing anticoagulant (Heparin-Na)and placed on wet ice. The plasma fraction was separated bycentrifugation and frozen at −20° C. for up to 4 h and −80° C. after 4 hunless analyzed shortly after sample collection. Colon samples werecollected at 4 h post dose. From the beginning of the cecum, a 4-6 cmsample of the colon was dissected, cut open on the longitudinal axis,and the solid contents removed by flushing with 2 mL of saline. Thecolon was further washed by putting it in 5 mL of saline and shaken for5 seconds. The colon sample was then patted dry, weighed, andhomogenized as 1 part tissue (g) to 4 parts HPLC grade water (mL).Concentrations of the compound in plasma and colon homogenate weredetermined using a qualified liquid chromatography-triple quadrupolemass spectrometry (LC-MS/MS) method. This protocol was used to evaluatethe following test compounds: Compounds (B) and (C) and Examples 6 and11.

Oral Dosing Protocol 2

Three non-fasted female C57BL/6 mice were orally administered testcompound at a dose of 25 mg/kg p.o. as a solution in 20% HPβCD at a dosevolume of 5 mL/kg. Blood samples were collected at 0.5, 2, and 4 h postdose via retro-orbital bleed or dorsal metatarsal vein. Blood sampleswere collected into tubes containing anticoagulant (Heparin-Na) andplaced on wet ice. The plasma fraction was separated by centrifugationand frozen at −20° C. for up to 4 h and −80° C. after 4 h unlessanalyzed shortly after sample collection. Colon samples were collectedat 4 h post dose. From 2 cm below the cecum, a 4 cm sample of the colonwas dissected, cut open on the longitudinal axis, and the solid contentsremoved by flushing with 2 mL of saline. The colon was further washed byputting it in 5 mL of saline and shaken for 5 seconds. The colon samplewas then patted dry, weighed, and homogenized as 1 part tissue (g) to 4parts HPLC grade water (mL). Concentrations of the compound in plasmaand colon homogenate were determined using a qualified liquidchromatography-triple quadrupole mass spectrometry (LC-MS/MS) method.This protocol was used to evaluate the following test compounds:Compound (A) and Examples 1-5, 7-10, and 12.

IC Dosing-Protocol 3

Intracolonic (IC) dose group: Following anesthesia with isoflurane byinhalation, three non-fasted female C57BL/6 mice were administered thecompound intracolonically through a small incision in the abdominal wallusing a syringe and needle at a dose of 5 mg/kg as a solution in 20%HPβCD at a dose volume of 1 mL/kg. Blood samples were collected at 0.5,2, and 4 h post dose via retro-orbital bleed. Blood samples werecollected into tubes containing anticoagulant (Heparin-Na) and placed onwet ice. The plasma fraction was separated by centrifugation and frozenat −20° C. for up to 4 h and −80° C. after 4 h unless analyzed shortlyafter sample collection. Colon samples were collected at 4 h post dose.From 2 cm below the cecum, a 4-cm sample of the colon was dissected, cutopen on the longitudinal axis, and the solid contents removed byflushing with 2 mL of saline. The colon was further washed by putting itin 5 mL of saline and shaken for 5 seconds. The colon sample was thenpatted dry, weighed, and homogenized as 1 part tissue (g) to 4 partsHPLC grade water (mL). Concentrations of the compound in plasma andcolon homogenate were determined using a qualified liquidchromatography-triple quadrupole mass spectrometry (LC-MS/MS) method.This protocol was used to evaluate IC dosing of the following testcompounds: Examples 1, 3, and 4.

Compounds Ex. 1-12 are further characterized by the physico-chemicalproperties given in Table 8. c Log P and tPSA values were calculated byusing ChemBioDraw Ultra 14.0, where P is the n-octanol—water partitioncoefficient. The total polar surface area (tPSA) is calculated as thesurface sum over all polar atoms, primarily oxygen and nitrogen, alsoincluding their attached hydrogens.

TABLE 8 Some physico-chemical properties of compounds Ex. 1-12 Test # Hbond # H bond # rotatable Compound cLog P tPSA donors acceptors bondsEx. 1 0.94 113.11 3 5 6 Ex. 2 2.31 88.17 2 4 3 Ex. 3 1.58 92.88 2 4 6Ex. 4 0.54 116.67 2 5 6 Ex. 5 0.24 102.11 2 5 5 Ex. 6 0.86 102.11 2 5 6Ex. 7 1.25 116.67 2 5 6 Ex. 8 1.13 113.11 3 5 6 Ex. 9 1.14 108.48 2 5 5Ex. 10 1.35 116.67 2 5 6 Ex. 11 1.50 117.27 3 5 5 Ex. 12 0.57 113.11 3 56

1-55. (canceled)
 56. A compound of formula